Methods of increasing response to cancer radiation therapy

ABSTRACT

Disclosed herein are methods of increasing response to radiation therapy in subjects afflicted with cancer. In some embodiments, the method comprises reducing the ability of an immune suppressor cell (e.g., MDSC) to migrate to the microenvironment of the cancer. In some embodiments, the method further comprises suppressing the migration of the immune suppressor cell to a non-malignant cell and/or suppressing the malignant transformation of the non-malignant cells.

This application is a divisional of U.S. application Ser. No.17/375,459, filed Jul. 14, 2021, which is a continuation of U.S.application Ser. No. 15/019,924, filed Feb. 9, 2016, which is (a) acontinuation-in-part of PCT International Application No.PCT/US2014/050442, filed on Aug. 8, 2014, claiming the benefit of U.S.Provisional Application No. 61/864,509, filed Aug. 9, 2013; (b) claimsthe benefit of U.S. Provisional Application No. 62/115,011, filed Feb.11, 2015; and (c) claims the benefit of U.S. Provisional Application No.62/220,138, filed Sep. 17, 2015, the content of each of the foregoingapplications is hereby incorporated by reference in its entirety.

This invention was made with government support under grant numbers RO1CA055306, RO1 CA149425, RO1 CA089481, T32 CA009140, and P30 CA016520awarded by the National Institutes of Health. The U.S. Government hascertain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING: The contents of theelectronic sequence listing (PENN 002_06US_SeqList_ST26.xml; Size:13,544 bytes; and Date of Creation: Aug. 8, 2022) is herein incorporatedby reference in its entirety.

Throughout this application, various publications are referenced,including referenced in parenthesis. Full citations for publicationsreferenced in parenthesis may be found listed at the end of thespecification immediately preceding the claims. The disclosures of allreferenced publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

BACKGROUND OF INVENTION

The erbB family of receptors includes erbB1 (EGFR), erbB2(p185her2/neu), erbB3 and erbB4. Ullrich, et al. (1984) Nature 309,418-425, which is incorporated herein by reference, describes EGFR.Schechter, A. L., et al. (1984) Nature 312, 513-516, and Yamamoto, T.,et al. (1986) Nature 319, 230-234, which are each incorporated herein byreference, describe p185neu/erbB2. Kraus, M. H., et al. (1989) Proc.Natl. Acad. Sci. USA 86, 9193-9197 which is incorporated herein byreference, describes erbB3. Plowman, G. D., (1993) Proc. Natl. Acad.Sci. USA 90, 1746-1750, which is incorporated herein by reference,describes erbB4.

The rat cellular protooncogene c-neu and its human counterpart c-erbB2encode 185 kDa transmembrane glycoproteins termed p185her2/neu. Tyrosinekinase (tk) activity has been linked to expression of the transformingphenotype of oncogenic p185her2/neu (Bargmann et al., Proc. Natl. Acad.Sci. USA, 1988, 85, 5394; and Stem et al., Mol. Cell. Biol., 1988, 8,3969, each of which is incorporated herein by reference). Oncogenic neuwas initially identified in rat neuroglioblastomas (Schechter et al.,Nature, 1984, 312, 513, which is incorporated herein by reference) andwas found to be activated by a carcinogen-induced point mutationgenerating a single amino acid substitution, a Val to Glu substitutionat position 664, in the transmembrane region of the transforming protein(Bargmann et al., Cell, 1986, 45, 649, which is incorporated herein byreference). This alteration results in constitutive activity of itsintrinsic kinase and in malignant transformation of cells (Bargmann etal., EMBO J., 1988, 7, 2043, which is incorporated herein by reference).The activation of the oncogenic p185her2/neu protein tyrosine kinaseappears to be related to a shift in the molecular equilibrium frommonomeric to dimeric forms (Weiner et al., Nature, 1989, 339, 230, whichis incorporated herein by reference).

Overexpression of c-neu or c-erbB2 to levels 100-fold higher than normal(i.e., >10⁶ receptors/cell) also results in the transformation of NIH3T3cells (Chazin et al., Oncogene, 1992, 7, 1859; DiFiore et al., Science,1987, 237, 178; and DiMarco et al., Mol. Cell. Biol., 1990, 10, 3247,each of Which is incorporated herein by reference). However, NIH3T3cells or NR6 cells which express cellular p185her2/neu at the level of10⁵ receptors/cell are not transformed (Hung et al., Proc. Natl. Acad.Sci. USA, 1989, 86, 2545; and Kokai et al., Cell, 1989, 58, 287, each ofwhich is incorporated herein by reference), unless co-expressed withepidermal growth factor receptor (EGFR), a homologous tyrosine kinase(Kokai et al, Cell, 1989, 58, 287, which is incorporated herein byreference). Thus, cellular p185her2/neu and oncogenic p185her2/neu mayboth result in the transformation of cells.

Cellular p185her2/neu is highly homologous with EGFR (Schechter et al.,Nature, 1984, 312, 513; and Yamamoto et al., Nature, 1986, 319, 230,each of which is incorporated herein by reference) but nonetheless isdistinct. Numerous studies indicate that EGFR and cellular p185her2/neuare able to interact (Stem et al., Mol. Cell. Biol., 1988, 8, 3969; Kinget al., EMBO J., 1988, 7, 1647; Kokai et al., Proc. Natl. Acad. Sci.USA, 1988, 85, 5389; and Dougall et al., J. Cell. Biochem., 1993, 53,61; each of which is incorporated herein by reference). Theintermolecular association of EGFR and cellular p185her2/neu appear toup-regulate EGFR function (Wada et al., Cell, 1990, 61, 1339, which isincorporated herein by reference). In addition, heterodimers which formactive kinase complexes both in vivo and in vitro can be detected (Qianet al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1330, which isincorporated herein by reference).

Similarly, p185her2/neu interactions with other erbB family members havebeen reported (Carraway et al., Cell 1994, 78, 5-8; Alroy et al., FEBSLett. 1997, 410, 83-86; Riese et al., Mol. Cell. Biol. 1995, 15,5770-5776; Tzahar et al., EMBO J. 1997, 16, 4938-4950; Surden et al.,Neuron 1997, 18, 847-855; Pinkas-Kramarski et al., Oncogene 1997, 15,2803-2815; each of which is incorporated herein by reference). Humanp185her2/neu forms heterodimers with either erbB3 or erbB4 underphysiologic conditions, primarily in cardiac muscle and the nervoussystem, particularly in development.

Cellular p185her2/neu proteins are found in adult secretory epithelialcells of the lung, salivary gland, breast, pancreas, ovary,gastrointestinal tract, and skin (Kokai et al., Proc. Natl. Acad. Sci.USA, 1987, 84, 8498; Mori et al., Lab. Invest, 1989, 61, 93; and Presset al., Oncogene, 1990, 5, 953; each of which is incorporated herein byreference). Recent studies have found that the amplification of c-erbB2occurs with high frequency in a number of human adenocarcinomas such asgastric (Akiyama et al., Science, 1986, 232, 1644, which is incorporatedherein by reference), lung (Kern et al., Cancer Res., 1990, 50, 5184,which is incorporated herein by reference) and pancreaticadenocarcinomas (Williams et al., Pathobiol, 1991, 59, 46, which isincorporated herein by reference). It has also been reported thatincreased c-erbB2 expression in a subset of breast and ovariancarcinomas is linked to a less optimistic clinical prognosis (Slamon etal., Science, 1987, 235, 177; and Slamon et al., Science, 1989, 244,707, each of which is incorporated herein by reference). Heterodimericassociation of EGFR and p185her2/neu has also been detected in humanbreast cancer cell lines, such as SK-Br-3 (Goldman et al., Biochemistry,1990, 29, 1 1024, which is incorporated herein by reference), andtransfected cells (Spivak-Kroizman et al., J. Biol. Chem, 1992, 267,8056, which is incorporated herein by reference). Additionally, cases oferbB2 and EGFR coexpression in cancers of the breast and prostate havebeen reported. In addition, heterodimeric association of p185her2/neuand erbB3 as well as heterodimeric association of p185her2/neu and erbB4have also been detected in human cancers. Coexpression of erbB2 anderbB3 has been observed in human breast cancers. Coexpression of EGFR,erbB2, and erbB3 has been seen in prostate carcinoma.

Amplification and/or alteration of the EGFr gene is frequently observedin glial tumor progression (Sugawa, et al. (1990) Proc. Natl. Acad. Sci.87: 8602-8606; Ekstrand, et al. (1992) Proc. Natl. Acad. Sci. 89:4309-4313), particularly in glioblastoma, the most malignant glial tumor(Libermann, et al. Supra; Wong, et al. Supra; James, et al. (1988)Cancer Res. 48: 5546-5551; Cavenee, W. K. (1992) Cancer 70:1788-93;Nishikawa, et al., (1994) Proc. Natl. Acad. Sci. 91: 7727 7731;Schlegel, et al. (1994) Int J. Cancer 56: 72-77). A significantproportion of these tumors show EGFr amplification with or without genealteration (Ekstrand, et al. Supra; Libermann, et al. Supra; Wong, etal. (1987) Proc. Natl. Acad. Sci. 84:6899-6903), and this has beencorrelated with a shorter interval to disease recurrence and poorersurvival (Schlegel, et al. Supra).

EGFr amplification can be associated with aberrant EGFr transcriptsalong with normal EGFr transcripts (Sugawa, et al. Supra). Frequentamplification and subsequent structural alteration suggests the EGFr maybe important for the maintenance of the phenotype of malignant glioma. Afrequently observed EGFr mutant has been identified in a subset of humanglioblastomas and results from an in-frame truncation of 801 bp(corresponding to exons 2-7) in the extracellular domain of the receptor(Sugawa, et al. Supra; Ekstrand, et al. Supra; Malden, et al. (1988)Cancer Res. 48: 2711-2714; Humphrey, et al. (1990) Proc. Natl. Acad.Sci. 87: 4207-4211; Wong, et al. (1992) Proc. Natl. Acad. Sci. 89:2965-2969), which is thought to result in constitutive kinase activationand may also affect the ligand-binding properties of the molecule(Nishikawa, et al. Supra; Callaghan, et al. (1993) Oncogene 8:2939-2948).

Observed mutations of EGFr in human epithelial malignancies consist ofoverexpression with or without amplification and, less commonly, ofcoding sequence alterations. Oncogenic transformation caused by mutantsof EGFr appear to be tissue-specific and have been observed in erythroidleukemia, fibrosarcoma, angiosarcoma, melanoma, as well as glioblastoma(Carter, et al. (1994) Crit Rev Oncogenesis 5:389-428). Overexpressionof the normal EGFr may cause oncogenic transformation in certain cases,probably in an EGF-dependent manner (Carter, et al. Supra; Haley, et al.(1989) Oncogene 4: 273-283). Transfection of high amounts of wild-typeEGFr into NIH3T3 cells results in ligand-dependent but incompletetransformation (YamaZaki, et al. (1990) Jpn. J. Cancer Res. 81:773-779). Overexpression may cause altered cell-cycle regulation of theEGFr kinase, and contribute to the transformed state, as has beenobserved for oncogenic p185neu (Kiyokawa, et al. (1995) Proc. Natl.Acad. Sci. 92:1092-1096).

There is a need for therapeutic compositions useful to treat individualsidentified as having erbB2/Her2/neu or EGFR-mediated tumors. There is aneed for methods of treating individuals identified as havingerbB2/Her2/neu or EGFR-mediated tumors.

SUMMARY OF THE INVENTION

The present invention provides a fusion protein comprising

(a) a first stretch of consecutive amino acids, the sequence of which isthe sequence of an anti-p185her2/neu polypeptide;

(b) a second stretch of consecutive amino acids, the sequence of whichis the sequence of a polypeptide capable of binding at least onepolypeptide other than p185her2/neu; and

(c) a third stretch of consecutive amino acids, the sequence of whichcomprises the sequence of a biologically active portion ofinterferon-gamma (IFNγ),

wherein (b) is located at the carboxy-terminal end of (a), and (c) islocated at the carboxy-terminal end of (b).

The present invention provides a method of treating a subject afflictedwith cancer, which comprises administering to the subject atherapeutically effective amount of a fusion protein of the invention.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of sensitizing cells of a cancerin a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention, and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention provides a method of treating a subject afflictedwith cancer or preventing the development of a tumor in a subject atrisk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an anti-p185her2/neu antibody whichinhibits p185her2/neu signaling in the cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and concurrently or subsequently administering IFNγ whichinduces further phenotypic change in the cancer cell; or

ii) administering to the subject a fusion protein of the invention,wherein the first stretch of consecutive amino acids of the fusionprotein inhibits p185her2/neu signaling in a cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and the third stretch of consecutive amino acids of the fusionprotein induces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

The present invention provides a composition for the treatment of asubject afflicted with cancer, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing cancer toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing a tumor toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a fusion protein comprising

(a) a first stretch of consecutive amino acids, the sequence of which isthe sequence of an anti-EGFR polypeptide;

(b) a second stretch of consecutive amino acids, the sequence of whichis the sequence of a polypeptide capable of binding at least onepolypeptide other than EGFR; and

(c) a third stretch of consecutive amino acids, the sequence of whichcomprises the sequence of a biologically active portion ofinterferon-gamma (IFNγ),

wherein (b) is located at the carboxy-terminal end of (a), and (c) islocated at the carboxy-terminal end of (b).

The present invention provides a method of treating a subject afflictedwith cancer, which comprises administering to the subject atherapeutically effective amount of the fusion protein of the invention.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of sensitizing cells of a cancerin a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention, and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention provides a method of treating a subject afflictedwith cancer or preventing the development of a tumor in a subject atrisk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an anti-EGFR antibody which inhibitsEGFR signaling in the cancer cell, wherein said inhibition converts thephenotype of the cancer cell such that the cancer cell is amenable tofurther phenotypic change by interferon-gamma (IFNγ), and concurrentlyor subsequently administering IFNγ which induces further phenotypicchange in the cancer cell; or

ii) administering to the subject a fusion protein of the invention,wherein the first stretch of consecutive amino acids of the fusionprotein inhibits EGFR signaling in a cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and the third stretch of consecutive amino acids of the fusionprotein induces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

The present invention provides a composition for the treatment of asubject afflicted with cancer, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing cancer toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing a tumor toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

The present invention provides a polynucleotide encoding a fusionprotein of the invention.

The present invention provides an expression vector comprising apolynucleotide of the invention operably linked to a promoter.

The present invention provides a cell comprising a expression vector ofthe invention.

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress p185her2/neu in a subjectin need of such inhibition which comprises administering to said subject

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ), each in asufficient amount to down regulate the overexpressed p185her2/neu andinhibit the development of said breast cells that overexpressp185her2/neu into breast cancer cells.

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress EGFR in a subject in needof such inhibition which comprises administering to said subject

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ), each in asufficient amount to down regulate the overexpressed p185 and inhibitthe development of said breast cells that overexpress p185 into breastcancer cells.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

The present invention also provides a method of sensitizing cells of acancer in a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

The present invention further provides a method of treating a subjectafflicted with cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ), and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention also provides a method of treating a subjectafflicted with cancer or preventing the development of a tumor in asubject at risk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an erbB inhibitor which inhibits EGFRsignaling or p185her2/neu signaling in the cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and concurrently or subsequently administering IFNγ whichinduces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

The present invention provides a method of treating a subject afflictedwith a tumor associated with EGFR or p185her2/neu or preventingdevelopment of a tumor associated with EGFR or p185her2/neu in asubject, which comprises administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress p185her2/neu in a subjectin need of such inhibition which comprises administering to said subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressedp185her2/neu and inhibit the development of said breast cells thatoverexpress p185her2/neu into breast cancer cells.

The present invention also provides a method of inhibiting developmentinto cancer cells of breast cells that overexpress EGFR in a subject inneed of such inhibition which comprises administering to said subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressed p185 andinhibit the development of said breast cells that overexpress p185 intobreast cancer cells.

Aspects of the present invention relate to a composition for thetreatment of a subject afflicted with cancer, comprising i) an erbBinhibitor; and ii) interferon-gamma (IFNγ), and a pharmaceuticallyacceptable carrier.

Aspects of the present invention also relate to a composition forsensitizing cancer to radiation or a chemotherapeutic agent, comprisingi) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention relate to a composition for preventingthe development of a tumor in a subject at risk of developing the tumor,comprising i) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention also relate to a composition forsensitizing a tumor to radiation or a chemotherapeutic agent, comprisingi) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention relate to a combination for thetreatment of a subject afflicted with cancer or preventing thedevelopment of a tumor in a subject at risk of developing the tumor,comprising i) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Enhanced in vivo activity of anti-Her2/neu antibody and IFNγ.H2N113 tumor cells (1×10⁶) were injected subcutaneously into both sideof the back of 6-10 weeks old MMTV-neu mice. Once tumors reached anaverage size of 30-40 mm³ (10-12 days after tumor inoculation), micewere treated with control IgG, IFN-γ (5×10⁵ IU/kg, three times perweek), 7.16.4 (1.5 mg/kg, twice per week), or the combination of IFN-γand 7.16.4. Data represent mean+SEM. t test was performed to compare thedifference in the tumor size of different treatment groups. * P<0.05,**p<0.01, compared with control; ^(#)P<0.05, ^(##)P<0.01, compared withthe 7.16.4 group; ^(&)P<0.05, ^(&&)P<0.01, compared with the IFN-γgroup.

FIG. 2A-FIG. 2B. In vivo activity of 4D5scFvZZ-IFNγ. FIG. 2A. Comparisonof 4D5scFvZZ (SEQ ID NO: 1) and 4D5scFvZZIFNγ. Tumors were palpable 5days after inoculation of transformed T6-17 cells. Mice were treatedwith control (PBS), 4D5 mAb (1 mg/kg, twice; then mg/kg twice, for atotal of 4 treatments in 2 weeks), 4D5scFvZZIFNγ (7 mg/kg, 5 times perweek), or 4D5scFvZZ (SEQ ID NO: 1) (7 mg/kg, 5 times per week). Tumorgrowth in the 4D5scFvZZIFNγ group was much suppressed compared withother groups. FIG. 2B. Dose-dependent activity of 4D5scFvZZIFNγ. Micewere treated with control (PBS), 4D5 mAb (1 mg/kg, twice per week), or4D5scFvZZIFNγ (7 mg/kg, or 1.75 mg/kg, 5 times per week). Tumor growthwas dose-dependently suppressed by 4D5scFvZZIFNγ.

FIG. 3 . 4D5scFv-ZZ-IFNγ and 4D5scFv-IFNγ bind to cell surfacep185her2/neu. T6-17 cells with the expression of p185her2/neu wereprepared for Fluorescence-activated cell sorting (FACS). Histogramsrepresent staining with 0.5 μg of 4D5scFv-IFNγ or 4D5scFv-ZZ-IFNγ, asindicated in the figure, followed by His-Probe antibody andAlexa488-conjugated goat anti-rabbit antibodies. The control stainingwas obtained with only the His-Probe antibody and the secondaryantibody.

FIG. 4 . Effect of 4D5scFv-ZZ-IFNγ on MHC expression. SKBR3 cells wereincubated with IFNγ or 4D5scFv-ZZ-IFNγ at different dose. Expression ofclass I and class II MHC antigens were analyzed by FACS using monoclonalantibodies W6/33 and L243, respectively.

FIG. 5 . Comparison of in vivo activity of 4D5scFv-ZZ-IFNγ and4D5scFv-IFNγ. 5×10⁵ T6-17 cells were injected s.c. into nude mice toinduce tumor growth. i.p. treatments with 4D5scFv (SEQ ID NO: 3) or4D5scFvZZ (SEQ ID NO: 1) were provided to mice (3 mg/kg/dose, 5 timesper week) started two days after inoculation. Error bars represent thestandard error of mean. * & **: The size of tumors was significantlydifferent from the controls (t test, *: p<0.05; **: p<0.01).

FIG. 6 . The enhanced activity of IFNγ and 7.16.4 can be seen in nudemice

FIG. 7 . IFNα appears not to have the same activity as IFNγ tofacilitate anti-Her2/neu antibody. IFNα appears to have anti-tumoractivity on its own in the in vivo tumor model but it could not enhancemAb 7.16.4 activity to suppress the growth of xenografted tumors.

FIG. 8 . Effect of co-treatment on MDSC.

FIG. 9 . Total cells migrated to the lower chamber.

FIG. 10 . IFN-7 and 4D5 exhibit enhanced activity on breast cancer cellsin vitro. SK-BR-3 cells were treated for eight days with IFN-γ (100IU/mL), 4D5 (10 μg/mL), or both. On the eighth day, cell viability wasmeasured by MTT assay. Preliminary experiments revealed that combinationof IFN-γ and 4D5 for the entire eight days was superior to pre-treatmentwith 4D5 for four days followed by addition of IFN-γ with 4D5 for thefinal four days or pre-treatment with IFN-γ followed by addition 4D5with IFN-γ for the final four days (the latter had no effect).

FIG. 11 . IFN-γ and 4D5-mediated Snail degradation is mediated throughthe proteasome. SK-BR-3 cells were treated with IFN-γ (100 IU/mL), 4D5(10 μg/mL), IFN-γ+4D5, or scFv4D5 (IFN-γ) (10 μg/mL) for 24 hours in thepresence or absence of the proteasome inhibitor, MG-132 (10 μM). Aftertreatment, cells were lysed and Snail content was assayed by Westernblot; β-actin was used as a loading control.

FIG. 12 . IFN-γ and 4D5-mediated Snail degradation requires GSK-3β.SK-BR-3 cells were treated with IFN-γ (10 ng/mL), 4D5 (10 μg/mL), orboth for 24 hours along with the indicated concentration of the GSK-3βinhibitor, CHIR99021. After treatment, cells were lysed and Snail andSlug content was assayed by Western blot; β-actin was used as a loadingcontrol.

FIG. 13 . IFN-γ and 4D5 decreases Snail Half-Life. SK-BR-3 cells weretreated as control or IFN-γ (100 IU/mL)+4D5 (10 μg/mL) in combinationwith the translation inhibitor, cycloheximide (CHX, 10 μg/mL) for theindicated times. After treatment, cells were lysed and Snail content wasassayed by Western blot; β-actin was used as a loading control. Ub-Snailindicates likely ubiquitinated forms of Snail.

FIG. 14 . Survivin Inhibition Response. SK-BR-3 cells were pretreatedwith IFN-γ (10 ng/mL), 4D5 (10 μg/mL), IFN-γ+4D5, or scFv4D5 (IFN-γ) (10μg/mL) for 24 hours followed by the addition of the Survivin inhibitor,S12 at the indicated doses. Forty-eight hours later, cell viability wasmeasured by MTT assay.

FIG. 15 . Ionizing Radiation Response. SK-BR-3 cells were pretreatedwith IFN-γ (10 ng/mL), 4D5 (10 μg/mL), IFN-γ+4D5, or scFv4D5 (IFN-γ) (10μg/mL) for 24 hours followed by exposure to ionizing radiation at theindicated doses. Forty-eight hours later, cell viability was measured byMTT assay.

FIG. 16 . A431Lx cells were seeded at 1,000 cells/well in a 96-wellplate and 24 hours later were treated with IFN-γ (10 ng/mL), C225 (alsoknown as cetuximab) (10 μg/mL), or both. Forty-eight hours later, cellviability was measured by MTT assay.

FIG. 17 . A431Lx cells were seeded at 2,500 cells/well in a 96-wellplate and 24 hours later were treated with IFN-γ (10 ng/mL), C225 (10μg/mL), or both. Forty-eight hours later, cell viability was measured byMTT assay.

FIG. 18 . A431Lx cells were seeded at 5,000 cells/well in a 96-wellplate and 24 hours later were treated with IFN-γ (10 ng/mL), C225 (10μg/mL), or both. Forty-eight hours later, cell viability was measured byMTT assay.

FIG. 19A-FIG. 19D. Anti-erbB2 monoclonal antibody and IFN-γ act directlyon HER2-positive breast cancer cells. (FIG. 19A) SK-BR-3 cells weretreated with regimens of anti-erbB2 mAb (4D5) and IFN-γ at the indicatedconcentrations. Treatments described as IFN-γ→D5+IFN-γ were treated withthe indicated dose of IFN-γ for 4 days, then the indicated dose of IFN-γplus 4D5 for an additional 4 days. Treatments described as 4D5→4D5+IFN-γwere treated with 4D5 for 4 days, then the indicated dose of IFN-γ plus4D5 for an additional 4 days. All other treatment groups were treated asindicated for 8 days. Following a total of 8 days, an MTT assay wasperformed. Data were normalized to the cIgG group. Bar graphs representthe mean±S.D. of a typical experiment (n=5). (FIG. 19B) SK-BR-3 cellswere treated as indicated and MTT assays were performed every secondday. Data were normalized to the cIgG group on Day 0. Data pointsrepresent the mean±S.D. of a typical experiment (n=6). (FIG. 19C)SK-BR-3 cells were seeded in a 0.2% agar solution containing theindicated treatments, which was layered over a 0.8% agar solution. After14 days in culture, viable foci were visualized following incubationwith MTT solution. Scale bar is 250 μm. (FIG. 19D) Foci from panel Cwere quantified using NIH-endorsed software (ImageJ). Bar graphsrepresent the mean±S.D. of a typical experiment (n=3 or 4; n.s., notsignificant; *, p<0.05; **, p<0.01; ***; p<0.001).

FIG. 20A-FIG. 20D. Disabling HER2 kinase activity principallyinactivates Akt signaling. (FIG. 20A) SK-BR-3 cells were treated withvehicle (0.001% DMSO), lapatinib, cIgG, 4D5, or C225 as indicated for 24hours. (FIG. 20B) After three days in culture, MDAMB-453 and SK-BR-3cells were treated with vehicle (0.001%) or indicated doses of lapatinibfor 24 hours. (FIG. 20C) SK-BR-3 cells were treated with vehicle (0.05%DMSO) or the PI-3K inhibitor LY294002 at the indicated dose for 24hours. (FIG. 20D) SK-BR-3 cells were treated with vehicle (0.01% DMSO)or the indicated dose of the Akt 1 and 2 inhibitor for 24 hours. In allinstances, equal amounts of lysate were separated by SDS-PAGE,transferred, and immunoblotted with the indicated antibodies. β-actin isused as a loading control. Shown are representative Western blots oftypical experiments.

FIG. 21A-FIG. 21D. Inclusion of IFN-γ potentiates the erbB2disabling-caused snail degradation. (FIG. 21A) SK-BR-3 cells weretreated for two days with cIgG, 4D5, or IFN-γ as indicated. (FIG. 21B)SK-BR-3 cells were treated for one day with cIgG, 4D5, or IFN-γ asindicated. The following day, nuclear and cytoplasmic fractions wereprepared from these cells. (FIG. 21C) SK-BR-3 cells were treated for twodays with cIgG, 4D5, C225, or IFN-γ as indicated. (FIG. 21D) SK-BR-3cells were treated for one day with vehicle (0.001% DMSO), three dosesof lapatinib, or IFN-γ (two doses) as indicated. In all instances, equalamounts of lysate were separated by SDS-PAGE, transferred, andimmunoblotted with the indicated antibodies. β-actin was used as aloading control. In panel B, Ku70 and phosphoinositide-dependentkinase-1 (PDK1) demonstrate enrichment for nuclear and cytoplasmicfractions, respectively. Shown are representative Western blots oftypical experiments.

FIG. 22A-FIG. 22C. IFN-γ, but not IFN-β, requires the inclusion ofanti-erbB2 mAb. (FIG. 22A) SK-BR-3 cells were treated for one day withcIgG, 4D5, IFN-β, or IFN-γ as indicated. Equal amounts of lysate wereseparated by SDS-PAGE, transferred, and immunoblotted with the indicatedantibodies. β-actin is used as a loading control. Shown is arepresentative Western blot of a typical experiment. (FIG. 22B) SK-BR-3cells were treated as indicated and MTT assays were performed everysecond day. Data were normalized to the cIgG group on Day 0. Data pointsrepresent the mean±S.D. of a typical experiment (n=6). (FIG. 22C) MCF10Acells were treated as indicated for 8 days. Vehicle control for IFN-γexperiments was performed by addition of media whereas vehicle controlfor IFN-β experiments was a vehicle (50 mM NaOAc, pH 5.5 containing 0.1%BSA). Following treatment, an MTT assay was used to assess viability.Data were normalized to IFN-γ control values. Bar graphs represent themean±S.D. of a typical experiment (n=5) (n.s., not significant; p<0.01;***; p<0.001).

FIG. 23A-FIG. 23C. Anti-erbB2 and IFN-γ degrade snail through theGSK3-β/proteasome pathway. SK-BR-3 cells were treated with cIgG, 4D5,and IFN-γ as indicated for two days. (FIG. 23A) During the final 8 hoursof treatment, vehicle (0.025% ethanol) or the indicated doses of theproteasome inhibitor MG-132 were added. (FIG. 23B) During the final 8hours of treatment, vehicle (0.01% DMSO) or the indicated doses of theGSK3-β inhibitor CHIR99021 were added. (FIG. 23C) SK-BR-3 cells weretransfected with either empty vector (EV), wild-type (WT) snail, orsnail with serines 97, 101, 108, 112, 116, and 120 mutated to alanines(6SA). The day following transfection, cytoplasmic and nuclear fractionswere prepared from these cells. In all instances, equal amounts oflysate were separated by SDS-PAGE, transferred, and immunoblotted withthe indicated antibodies. β-actin and Ku70 (nuclear fraction) were usedas loading controls. Shown are representative Western blots of typicalexperiments.

FIG. 24 . Graphical Abstract. In transformed cells, EGFR (purple andgray) and erbB2 (light and dark blue) can exist as heterodimers orhomodimers. These receptors activate the Ras/Raf/MEK/Erk and PI-3K/Aktpathways. Upon treatment with anti-erbB2 mAb, the Akt pathway becomesmoderately inactivated. Upon addition of IFN-γ to the mAb-disabledcells, the Akt pathway is further inactivated and snail is degradedthrough activated (nonphosphorylated GSK3-β).

FIG. 25 . MCF10A cells are apathetic to anti-erbB2 mAb and IFN-γtreatments. MCF10A cells were treated as indicated for 8 days. After 8days, an MTT assay was performed. Data were normalized to the cIgGgroup. Bar graphs represent the mean±S.D. of a typical experiment (n=6).MCF10A cells were treated with the MEK1/2 inhibitor U0126 as a killcontrol.

FIG. 26 . Surprising combined effects of anti-neu/p185 mAb therapycombined with IFN-γ in vivo. H2N113 tumor cells were injectedsubcutaneously into both sides of the back of BALB/c mice. After tumorsreached an average size of 30-40 mm³, mice were treated with control(PBS), IFN-γ (5×10⁵IU/kg), 7.16.4 (1.5 mg/kg), or IFN-γ+7.16.4. Datarepresent mean+SEM (* P<0.05, **P<0.01, compared with control; #P<0.05,##P<0.01, compared with the 7.16.4 group; & P<0.05, && P<0.01, comparedwith the IFN-γ group).

FIG. 27A-FIG. 27B. Dose-response effects of IFN-γ in the presence andabsence of mAb. A431 (FIG. 27A) and U87 (FIG. 27B) cells were treated asindicated for 8 days. After 8 days, an MTT assay was performed. Datawere normalized to the cIgG group. Bar graphs represent the mean±S.D. ofa typical experiment (n=6).

FIG. 28 . Anti-erbB2 and IFN-γ degrade snail independently of thelysosome. SK-BR-3 cells were treated with cIgG, 4D5, and IFN-γ asindicated for two days. During the final 8 hours of treatment, vehicle(ddH₂O) or the indicated doses of the lysosome inhibitor Chloroquinewere added. Equal amounts of lysate were separated by SDS-PAGE,transferred, and immunoblotted with the indicated antibodies. β-actinwas used as a loading control. Shown are representative Western blots ofa typical experiment.

FIG. 29 . Analysis of cell lines encompassing each subtype of breastcancer. Cell lines that model each classification of breast cancer wereharvested following three days in culture. Equal amounts of lysate wereseparated by SDS-PAGE, transferred, and immunoblotted with the indicatedantibodies. β-actin was used as a loading control. Shown arerepresentative Western blots of a typical experiment.

FIG. 30A-FIG. 30B. CD8+DC populations and cytotoxicity of CD8+ T cellsfrom spleen. FIG. 30A. Cells were gated on CD11c+MHC Class II+ cellswithout MDSC (CD11b^(high) and Gr-1^(high) cells). CD8+DEC205+ cellpopulations were shown as a percentage of CD11c+MHC class II+ cells.FIG. 30B: CD8+ T cells were collected from mouse spleens treated withcontrol IgG, 7.16.4+7.9.5, IFN-γ, and 7.16.4+7.9.5+IFN-γ. Cytotoxicityassays of the collected T cells were performed as described. Statisticalsignificance was calculated by the Student's t test.

FIG. 31 . IFN-γ improves two-antibody effect on the prevention of tumordevelopment. H2N113 tumor cells (0.25×10⁶) were injected s.c. intoMMTVneu mice at both flanks on day 0. Treatment for mice started atday 1. The doses for each agent included in the treatment were: 3.75μg/mouse for 7.16.4, 12.5 μg/mouse for 7.9.5, and 1×10⁴ IU/mouse forIFN-γ. Treatments were performed following a twice per week forantibodies and three times per week for αγ. t test indicated the tumorfree survival of the “7.16.4+7.9.5+IFN-γ group” is very significantlydifferent from the control group (P<0.001).

FIG. 32A-FIG. 32C. Establishment of IFNγR KD cells. FIG. 32A: Expressionlevel of IFNγR1 in H2N113 transfected with empty vector and shRNA (E3).FIG. 32B: Cells were stimulated with indicated concentration of IFN-γfor 16 h and MHC class I expression was assessed by flow cytometry withthe anti-H2Dd antibody. FIG. 32C: Cells were treated with indicatedconcentration of IFN-γ for 5 days before relative cell numbers weredetermined by the LDH activity of cell lysates.**P<0.01, ***P<0.005

FIG. 33 . Combination activity of anti-erbB2/neu antibody and IFN-γ isdependent on the IFN-γ receptor in the tumor cells. IFN-γ receptor wasknocked down by shRNA in H2N113. H2N113 cells were infected with emptyor shRNA-containing lentivirus and selected with 1 μg/ml puromycin.IFγR1 knockdown was confirmed by FACS analysis using anti-IFγR1 antibodyand by analyzing their expression of MHC class I following stimulationwith IFN-γ and proliferation (FIG. 32A-FIG. 32C). The resulting tumorcells (1×10⁶) were injected subcutaneously into MMTV-neu mice andtreated similarly as in FIG. 1 . Once tumors reached an average size of30-40 mm³ (10-12 days after tumor inoculation), mice were treated withPBS, IFN-γ (5×10⁵ IU/kg, three times per week), 7.16.4 (1.5 mg/kg, twiceper week), or the combination of IFN-γ and 7.16.4. Data representmean+SEM. t test was performed to compare the difference in the tumorsize of different treatment groups. * P<0.05, **P<0.01, compared withcontrol; ^(#)P<0.05, ^(##)P<0.01, compared with the 7.16.4 group;^(&)P<0.05, ^(&&)P<0.01, compared with the IFN-γ group.

FIG. 34 . IFN-γ further enhances the activity of anti-erbB2/neu antibodyand chemotherapy. H2N113 tumor cells (1×10⁶) were injectedsubcutaneously into both side of the back of 6-10 weeks old MMTV-neumice. Once tumors reached an average size of 30-40 mm³ (10-12 days aftertumor inoculation), mice were treated with control, 7.16.4 (1.5 mg/kg,twice per week)+docetaxel (5.5 mg/kg, twice per week), or7.16.4+docetaxel+IFN-γ (5×10⁵ IU/kg, three times per week). Datarepresent mean+SEM. t test was performed to compare the difference inthe tumor size of different treatment groups. * P<0.05, **P<0.01,***P<0.001, compared with control; ^(#)P<0.05, ^(##)P<0.01, comparedwith the 7.16.4+docetaxel group.

FIG. 35 . Effect of anti-PD1 antibody on IFN-γ and anti-erbB2/neuantibody. H2N113 tumor cells (1×10⁶) were injected subcutaneously intoboth side of the back of 6-10 weeks old MMTV-neu mice. Once tumorsreached an average size of 30-40 mm³ (10-12 days after tumorinoculation), mice were treated with control, 7.16.4 (1.5 mg/kg, twiceper week)+IFN-γ (5×10⁵ IU/kg, three times per week), or7.16.4+IFN-γ+anti-PD1 antibody (5 mg/kg, twice per week). Data representmean+SEM. t test was performed to compare the difference in the tumorsize of different treatment groups. * P<0.05, **P<0.01, ***P<0.001,compared with control.

FIG. 36 . In vivo activity of 4D5scFvZZ-mIFN-γ. T6-17 tumor cells(5×10⁵) were injected subcutaneously into both side of the back of 6-10weeks nude mice. The next day, mice were treated with control, 4D5(0.125 mg/kg) or 4D5scFvZZ-mIFN-γ (0.125 mg/kg), five times per week.Data represent mean+SEM. t test was performed to compare the differencein the tumor size of different treatment groups. * P<0.05, **P<0.01,***P<0.001, compared with control.

FIG. 37A-FIG. 37B. In vivo activity of 4D5scFvZZ-mIFN-γ is dependent onIFN-γ receptor. T6-17 (Vector; as shown in FIG. 37A) or T6-17 (IFN-γRKD; as shown in FIG. 37B) tumor cells (5×10⁵) were injectedsubcutaneously into the back of 6-10 weeks nude mice. The next day, micewere treated with control, 4D5 (0.125 mg/kg) or 4D5scFvZZ-mIFN-γ (0.125mg/kg), five times per week. Data represent mean+SEM.

FIG. 38A-FIG. 38B. Effect of co-treatment on MDSC cells. FIG. 38A.H2N113 tumors from each group of mice treated as indicated were obtainedafter treatment for FIG. 1 was finished. Tumor tissue was minced anddigested with collagenase P for 1 hr, followed by incubation withdispase and DNase for 5 minutes. Tumor-infiltrated MDSC cells wereisolated and compared using CD11b, Gr-1 and CD45 antibody by FACS. *P<0.05, * P<0.01, as compared with the IgG treated group. FIG. 38B. Invitro migration assay. H2N113 cells were seeded on 12-well plate andcultured until sub-confluent. Cells were then treated as indicated andconditioned media were collected at day 3 of culture. Migration of MDSCwas measured by the Transwell system (pore size: 4 μm). MDSCs wereisolated from spleens of tumor-bearing mice using MACS MDSC isolationkit, then seeded in the apical chamber. Condition media was then placedin the basolateral chamber and incubated for 3 hr. The cells thatmigrated to bottom chamber were collected and analyzed by flowcytometry. Fresh medium containing treatment reagents were used ascontrols. #. P<0.05 (compared with either 7.16.4 or IFN-γ treated group.

FIG. 39 . In vivo co-treatment significantly reduced the expression ofALDH1 in the tumor. Tumors from each group of mice treated as indicatedwere obtained after treatment for FIG. 1 was finished. Tumor tissue waslysed with modified RIPA buffer containing proteinase inhibitorcocktail. Each lysate was adjusted to 10 μg/lane and examined for Snailand ALDH1 expression by Western blot. β-actin was used as the loadingcontrol. Shown are representative Western blots of typical experiments.

FIG. 40 . H&E staining of tumors. Tumors were resected at the day afterfinal treatment and fixed with 10% buffered formalin, and subjected toH&E staining. Tumors from mice treated with 7.16.4 and IFN-γ showshigher necrosis. Arrows show necrotic area.

FIG. 41A-FIG. 41C. In vivo co-treatment led to increased M1 macrophageand reduced M2 macrophages in the tumors. Tumors from mice treated asindicated were obtained after treatment for FIG. 33 was finished. Tumorinfiltrated macrophages were examined by FACS. Accumulation of M1macrophages is shown in FIG. 41A; accumulation of M2 macrophages isshown in FIG. 41B; and the ratio of M1 macrophages to M2 macrophages ineach of the treatment conditions is shown in FIG. 41C. *P<0.05.

FIG. 42 . Treatment with mAb 4D5 (Herceptin) that targets the erbB2ectodomain, followed by IFN-γ, limits tumor growth. Interestingly, whenthe treatment is coupled with paclitaxel, greater tumor death was noted.In this growth assay we see doubling of the paclitaxel effectivenesswhen coupled with mAb and IFN-γ therapy using a standard MTT assay.Effective inhibition is seen with a much lower dose of paclitaxel. Thispreliminary study clearly indicates it is possible to reduce thegenotoxic drug amount when coupled with targeted mAb therapy followed byIFN-γ.

FIG. 43A-FIG. 43D. Effects of mAb therapy combined with IFN-γ in vitro.Different cell types (MDA-MB-453 in FIG. 43A; BT-474 in FIG. 43B;MDA-MB-231 in FIG. 43C; and A431 in FIG. 43D) were treated as indicatedand MTT assays were performed every second day. Data were normalized tothe cIgG group on Day 0. Data points represent the mean±S.D. of atypical experiment (n=6).

FIG. 44 . Snail and KLF4 expressions in GSK3-β knock down SK-BR-3.SK-BR-3 cells were stably transfected with either control or GSK3-βsilencing short hairpin RNAs. Cells were treated for two days with cIgG,4D5 or IFN-γ as indicated. In each instance, cIgG concentration was 10μg/ml, 4D5 concentration was 10 μg/ml, and IFN-γ concentration was 10ng/ml. In all instances, equal amounts of lysate were separated bySDS-PAGE, transferred, and immunoblotted with the indicated antibodies.β-Actin was used as a loading control. Shown are representative Westernblots of typical experiments.

FIG. 45A-FIG. 45B. FIG. 45A. H2N113 tumors from each group of micetreated as indicated were obtained after treatment for FIG. 8A wasfinished. Tumor-infiltrated MDSC cells were isolated and compared usingCD11b, Gr-1 and CD45 antibody by FACS. * P<0.05, ** P<0.01, as comparedwith the IgG treated group. FIG. 45B. In vitro migration assay. H2N113cells were seeded on 12-well plate and cultured until sub-confluent.Cells were then treated with control IgG (10 μg/ml), 7.16.4 (10 μg/ml),INF-γ (10 IU/ml), and 7.16.4 and INF-γ. and conditioned media werecollected at day 3 of culture. Migration of MDSC was measured by theTranswell system (pore size: 4 μm). MDSCs were isolated from spleens oftumor-bearing mice, then seeded in the apical chamber. Condition mediawas then placed in the basolateral chamber and incubated for 3 hr. Thecells that migrated to bottom chamber were collected and analyzed byFACS. Fresh medium was used as controls (medium). #. P<0.05 (comparedwith either 7.16.4 or IFN-γ treated group.

FIG. 46 . H2N113 tumor cells (1×106) were injected subcutaneously intoMMTV-neu mice similarly as in A. Mice were treated with control PBS,7.16.4 (1.5 mg/kg, twice per week) and docetaxel (5.5 mg/kg, twice perweek), 7.16.4 and IFN-γ (5×105 IU/kg, three times per week), or thecombination of IFN-γ, 7.16.4 and docetaxel. Data represent mean+SEM. Astudent t-test was performed to compare the difference in the tumor sizeof different treatment groups. * P<0.05, **P<0.01, ***P<0.001,****P<0.0001 compared with control; #P<0.05, ##P<0.01, compared with the7.16.4 and docetaxel group; & P<0.05, && P<0.01, compared with the7.16.4 and IFN-γ group.

FIG. 47 . Tumor free survival: Survival proportions. Treatments wereStarted at 6 weeks of age. Antibody treatment: I.P. injection at 10ug/mouse, twice/week. IFN-γtreatment: 1,000 IU/mouse, twice/week.

Mice are genetically programmed to develop breast cancers in astochastic manner. The actual development of tumors from tissueactivated by the neu gene is used. An MMTV neu promotor is used. Thismodel is described in U.S. Pat. No. 6,733,752, issued May 11, 2004, theentire content of which is hereby incorporated herein by reference.

FIG. 48 . Inclusion of tamoxifen does not permit IFN-γ to accentuateanti-erbB2 mAb. BT-474 cells were treated as indicated for 8 days. After8 days, an MTT assay was performed. Data were normalized to the cIgGgroup. Bar graphs represent the mean±S.D. of a typical experiment (n=6).

FIG. 49 . U87 cells were not affected by the treatment. U87 cells weretreated as indicated and MTT assays were performed every second day.Data were normalized to the cIgG group on Day 0. Data points representthe mean±S.D. of a typical experiment (n=6).

FIG. 50 . Inclusion of IFN-γ potentiates the erbB2 disabling-causedsnail and slug degradation. SK-BR-3 cells were treated for three dayswith cIgG, 4D5, or IFN-γ as indicated. Equal amount of lysate wereseparated by SDS-PAGE, transferred, and immunoblotted with the indicatedantibodies. β-actin was used as a loading control. Shown arerepresentative Western blots of a typical experiment.

FIG. 51 . Knock down of Snail enhances the effect of 4D5 on SK-BR-3proliferation. SK-BR-3 cells were treated with or without 4D5 mAb (5μg/ml). After 6 days, MTT assays were performed. Data points representthe mean±S.D.

FIG. 52 . Tumor-specific cytotoxicity of CD8+ T cells in mouse spleens.CD8+ T cells were collected from the spleens of mice treated withcontrol IgG (IgG), 7.16.4, IFN-γ, and 7.16.4 and IFN-γ, then subjectedto cytotoxicity assay. Statistical analysis was performed using aStudent's t-test. **P<0.02 compared to control.

FIG. 53 . MDA-MB-231 cells (Triple negative breast cancer cell line)were treated with Lapatinib and INF-γ as indicated concentrations. After6 days, MTT assays were performed. Data points represent the mean±SD.

FIG. 54A-FIG. 54D. Anti-erbB2 monoclonal antibody and IFN-γ act directlyon HER2-positive breast cancer cells. FIG. 54A—SK-BR-3 cells weretreated with regimens of anti-erbB2 mAb (4D5) and IFN-γ at the indicatedconcentrations. Protocols described as IFN-γ 4D5+IFN-γ were treated withthe indicated dose of IFN-γ for 4 days, then the indicated dose of IFN-γplus 4D5 for an additional 4 days. Protocols described as 4D5→4D5+IFN-γwere treated with 4D5 for 4 days, then the indicated dose of IFN-γ plus4D5 for an additional 4 days. All other treatment groups were treated asindicated for 8 days. Following a total of 8 days, an MTT assay wasperformed. Data were normalized to the cIgG group. Bar graphs representthe mean±S.D. of a typical experiment (n=5). FIG. 54B SK-BR-3 cells weretreated as indicated and MTT assays were performed every second day.Data were normalized to the cIgG group on Day 0. Data points representthe mean±S.D. of a typical experiment (n=6). FIG. 54C—SK-BR-3 cells wereseeded in a 0.2% agar solution containing the indicated treatments,which was layered over a 0.8% agar solution. After 14 days in culture,viable foci were visualized following incubation with MTT solution. FIG.54D—Foci from panel C were quantified using NIHendorsed software(ImageJ). Bar graphs represent the mean±S.D. of a typical experiment(n=3 or 4; n.s., not significant; *, p<0.05; **, p<0.01; ***; p<0.001).

FIG. 55A-FIG. 55D. Effects of mAb therapy combined with IFN-γ in vitro.Different cell types (A431 in FIG. 55A; MDA-MB-453 in FIG. 55B;MDA-MB-231 in FIG. 55C; and BT-474 in FIG. 55D) were treated asindicated and MTT assays were performed every second day. Data werenormalized to the cIgG group on Day 0. Data points represent themean±S.D. of a typical experiment (n=6).

FIG. 56A-FIG. 56C. Inclusion of IFN-γ potentiates the erbB2disabling-caused Snail degradation. FIG. 56A—SK-BR-3 cells were treatedfor 48 hours with cIgG, 4D5, or IFN-γ as indicated and subjected towestern blotting. FIG. 56B—SK-BR-3 cells were treated for two days withcIgG, 4D5, C225, or IFN-γ as indicated, and the expression of Snail andSlug were detected by western blotting. FIG. 56C—SK-BR-3 cells weretreated for 24 hours with vehicle (0.001% DMSO), three doses oflapatinib, or IFN-γ (two doses) as indicated. In all instances, equalamounts of lysate were separated by SDS-PAGE, transferred, andimmunoblotted with the indicated antibodies. β-actin is used as aloading control.

FIG. 57A-FIG. 57C. IFN-γ, but not IFN-β, requires the inclusion ofanti-erbB2 mAb. FIG. 57A—SK-BR-3 cells were treated for one day withcIgG, 4D5, IFN-β, or IFN-γ as indicated. Equal amounts of lysate wereseparated by SDS-PAGE, transferred, and immunoblotted with the indicatedantibodies. β-actin is used as a loading control. Shown is arepresentative Western blot of a typical experiment. FIG. 57B—MCF10Acells were treated as indicated for 8 days. Vehicle control for IFN-γexperiments was performed by addition of media whereas vehicle controlfor IFN-β experiments was a vehicle (50 mM NaOAc, pH 5.5 containing 0.1%BSA). Following treatment, an MTT assay was used to assess viability.Data were normalized to IFN-γ control values. Bar graphs represent themean±S.D. of a typical experiment (n=5) (n.s., not significant; **,p<0.01; ***; p<0.001). FIG. 57C SKBR-3 cells were treated as indicatedand MTT assays were performed every second day. Data were normalized tothe cIgG group on Day 0. Data points represent the mean±S.D. of atypical experiment (n=6).

FIG. 58A-FIG. 58C. Anti-erbB2 and IFN-γ degrade Snail through theGSK3-β/proteasome pathway. SK-BR-3 cells were treated with cIgG, 4D5,and IFN-γ as indicated for two days. FIG. 58A—During the final 8 hoursof treatment, vehicle (0.01% DMSO) or the indicated doses of the GSK3-βinhibitor CHIR99021 were added. FIG. 58B—During the final 8 hours oftreatment, vehicle (0.025% ethanol) or the indicated doses of theproteasome inhibitor MG-132 were added. FIG. 58C—SK-BR-3 cells weretransfected with either empty vector (EV), wild-type (WT) Snail, orSnail with serines 97, 101, 108, 112, 116, and 120 mutated to alanines(6SA). The day following transfection, cytoplasmic and nuclear fractionswere prepared from these cells. In all instances, equal amounts oflysate were separated by SDS-PAGE, transferred, and immunoblotted withthe indicated antibodies. β-actin and Ku70 (nuclear fraction) were usedas loading controls. Shown are representative Western blots of typicalexperiments.

FIG. 59A-FIG. 59G. Synergistic activity of anti-erbB2/neu antibody andIFN-γ. FIG. 59A—Implanted H2N113 tumors were treated with control IgG,IFN-γ (three times per week), 7.16.4 (twice per week), or thecombination of IFN-γ and 7.16.4. FIG. 59B—IFN-γ receptor was knockeddown by shRNA in H2N113. The resulting tumor cells (1×106) were injectedsubcutaneously into MMTV-neu mice and treated similarly as in A. Datarepresent mean+SEM. A student's t-test was performed to compare thedifference in the tumor size of different treatment groups. * P<0.05,**P<0.01, compared with control; #P<0.05, ##P<0.01, compared with the7.16.4 group; & P<0.05, && P<0.01, compared with the IFN-γ group. FIG.59C—H2N113 tumors from each group of mice treated as indicated wereobtained after treatment for FIG. 59A was finished. Tumor-infiltratedMDSC cells were isolated and compared using CD11b, Gr-1 and CD45antibody by FACS. * P<0.05, ** P<0.01, as compared with the IgG treatedgroup. FIG. 59D—In vitro migration assay. H2N113 cells were seeded on12-well plate and cultured until sub-confluent. Cells were then treatedwith control IgG (10 μg/ml), 7.16.4 (10 μg/ml), INF-γ (10 IU/ml), and7.16.4 and INF-γ. and conditioned media were collected at day 3 ofculture. Migration of MDSC was measured by the Transwell system (poresize: 4 μm). MDSCs were isolated from spleens of tumor-bearing mice,then seeded in the apical chamber. Conditioned media were then placed inthe basolateral chamber and incubated for 3 hr. The cells that migratedto the bottom chamber were collected and counted. Fresh medium was usedas control (medium). #. P<0.05 (compared with either 7.16.4 or IFN-γtreated group). FIG. 59E—In vivo cotreatment significantly reduced theexpression of ALDH1 in the tumor. Tumors from each group of mice treatedas indicated were obtained after treatment for FIG. 59A was finished.Each lysate was adjusted to 10 μg/lane and examined for Snail and ALDH1expression by Western blot. β-actin was used as the loading control.Shown are representative Western blots of typical experiments. FIG.59F—H2N113 tumor cells (0.25×106) were injected subcutaneously into bothside of the back of 6-10 weeks old MMTV-neu mice at day 0. Mice weretreated with control PBS, 7.9.5 and 7.16.4 (0.625 mg/kg each), or thecombination of IFN-γ, 7.9.5 and 7.16.4 twice per week from day 1. FIG.59G—H2N113 tumor cells (1×106) were injected subcutaneously intoMMTV-neu mice similarly as in A. Mice were treated with control PBS,7.16.4 (1.5 mg/kg, twice per week) and docetaxel (5.5 mg/kg, twice perweek), 7.16.4 and IFN-γ (5×105 IU/kg, three times per week), or thecombination of IFN-γ, 7.16.4 and docetaxel. Data represent mean+SEM. Astudent t-test was performed to compare the difference in the tumor sizeof different treatment groups. * P<0.05, **P<0.01, ***P<0.001,****P<0.0001 compared with control; #P<0.05, ##P<0.01, compared with the7.16.4 and docetaxel group; & P<0.05, && P<0.01, compared with the7.16.4 and IFN-γ group.

FIG. 60A-FIG. 60C. PD-L1 expression is increased in tumors treated withIFN-γ. FIG. 60A—Western blotting was performed with anti-PD-L1 antibodywith the tumors from each indicated group of mice after treatment in thesame way as FIG. 59A. FIG. 60B—FACS analysis of PD-L1 expression intumor cells from control IgG and 7.16.4+IFN-γ treated mice. Tumor cellsare gated as CD45− large size cells which we confirmed most of them aretumor cells by 7.16.4 antibody. **P<0.02. FIG. 60C—Administration ofanti-PD-L1 antibody with the ordered therapy. Mice were treated withcontrol PBS, 7.16.4 (1.5 mg/kg, three times per week) and anti-PDL1 (5mg/kg, twice per week), 7.16.4 and IFN-γ (5×105 IU/kg, three times perweek), or the combination of IFN-γ, 7.16.4 and anti-PD-L1. Datarepresent mean+SEM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fusion protein comprising

(a) a first stretch of consecutive amino acids, the sequence of which isthe sequence of an anti-p185her2/neu polypeptide;

(b) a second stretch of consecutive amino acids, the sequence of whichis the sequence of a polypeptide capable of binding at least onepolypeptide other than p185her2/neu; and

(c) a third stretch of consecutive amino acids, the sequence of whichcomprises the sequence of a biologically active portion ofinterferon-gamma (IFNγ),

wherein (b) is located at the carboxy-terminal end of (a), and (c) islocated at the carboxy-terminal end of (b).

In some embodiments, the fusion protein further comprises anoligopeptide linker between (a) and (b).

In some embodiments, the fusion protein further comprises anoligopeptide linker between (b) and (c).

In some embodiments, the fusion protein further comprises a secondoligopeptide linker between (b) and (c).

In some embodiments, the amino acid sequence of the oligopeptide linkeris identical to the amino acid sequence of the second oligopeptidelinker.

In some embodiments, the amino acid sequence of the oligopeptide linkeris different from the amino acid sequence of the second oligopeptidelinker.

In some embodiments,

i) the oligopeptide is a polyglycine oligopeptide linker or aglycine-serine oligopeptide linker; and

ii) the second oligopeptide is independently a polyglycine oligopeptidelinker or a glycine-serine oligopeptide linker.

In some embodiments, the amino acid sequence of the polyglycineoligopeptide linker comprises at least two, three, four, five, six,seven, eight, nine, or ten, consecutive glycine residues.

In some embodiments, the amino acid sequence of the glycine-serineoligopeptide linker comprises at least two, three, four, five, six,seven, eight, nine, or ten, consecutive glycine residues.

In some embodiments, the C-terminal residue of the glycine-serineoligopeptide linker is a serine residue.

In some embodiments, (a) is directly contiguous with (b).

In some embodiments, (b) is directly contiguous with (c).

In some embodiments, the anti-p185her2/neu polypeptide is a chain of anantibody or a portion thereof.

In some embodiments, the antibody chain is a single chain variablefragment (scFv).

In some embodiments, the antibody chain is a monoclonal antibody chain.

In some embodiments, the monoclonal antibody chain is a human monoclonalantibody chain, a humanized monoclonal antibody chain, or a chimericantibody chain.

In some embodiments, the monoclonal antibody chain is a chimericantibody chain, and wherein a portion of the chimeric antibody chain isderived from a human antibody chain.

In some embodiments, the antibody is an anti-p185her2/neu antibody.

In some embodiments, the anti-p185her2/neu antibody is 4D5.

In some embodiments, the anti-p185her2/neu antibody is pertuzumab.

In some embodiments, the anti-p185her2/neu antibody is a trastuzumab.

In some embodiments, the anti-p185her2/neu antibody binds at least aportion of the same epitope as trastuzumab.

In some embodiments, the anti-p185her2/neu antibody is 7.16.4.

In some embodiments, the anti-p185her2/neu antibody is 7.9.5.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than p185her2/neu is capable of binding at least oneantibody.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than p185her2/neu is capable of binding at least oneantibody Fc-region.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than p185her2/neu is derived from a portion of ProteinA or Protein G that is capable of binding at least one antibodyFc-region.

In some embodiments, the least one antibody is endogenously expressed ina mammal.

In some embodiments, the sequence of the third stretch of consecutiveamino acids is identical to the sequence of IFNγ (SEQ ID NO: 5).

In some embodiments, the sequence of the fusion protein has beenmodified to reduce immunogenicity in a human.

In some embodiments, (a), (b), or (c) has been humanized.

In some embodiments, each of (a), (b), and (c) has been humanized.

In some embodiments, the sequence of each of (a), (b), and (c) is foundin an endogenous human polypeptide.

In some embodiments, the fusion protein is 4D5scFvZZ-IFNγ, or ahumanized derivative thereof.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises administering to the subject atherapeutically effective amount of a fusion protein of the invention.

In some embodiments, the method further comprises administering anantibody to the subject.

In some embodiments, the antibody is an anti-p185her2/neu antibody.

In some embodiments, the antibody is an anti-EGFR antibody.

In some embodiments, the anti-EGFR antibody is cetuximab.

In some embodiments, the antibody is an anti-PD1 or an anti-PD-L1antibody.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the method further comprises administering achemotherapeutic agent to the subject.

In some embodiments, the chemotherapeutic agent is administered to thesubject in an amount that is less than the amount that would beeffective to treat the subject if the chemotherapeutic agent wasadministered without the fusion protein.

In some embodiments, the method further comprises comprisingadministering a radiation to the subject.

In some embodiments, the radiation is administered to the subject in anamount that is less than the amount that would be effective to treat thesubject if the radiation was administered without the fusion protein.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of sensitizing cells of a cancerin a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention, and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing the cancer to radiation or a chemotherapeutic agent byadministering to the subject

i) an anti-p185her2/neu antibody which inhibits p185her2/neu signalingin a cancer cell, wherein said inhibition induces a cytostatic phenotypein the cancer cell, and interferon-gamma (IFNγ) which induces aphenotype in the cancer cell or in a non-malignant cell in the subject;or

ii) a fusion protein of the invention, wherein the first stretch ofconsecutive amino acids of the fusion protein inhibits p185her2/neusignaling in a cancer cell, said inhibition having a cytostatic effecton the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing the cancer to radiation or a chemotherapeutic agent byadministering to the subject

i) an anti-p185her2/neu antibody which inhibits p185her2/neu signalingin a cancer cell, wherein said inhibition induces a phenotype in thecancer cell, and interferon-gamma (IFNγ) which induces a furtherphenotype in the cancer cell or in a non-malignant cell in the subject;or

ii) a fusion protein of the invention, wherein the first stretch ofconsecutive amino acids of the fusion protein inhibits p185her2/neusignaling in a cancer cell, wherein said inhibition induces a cytostaticphenotype in the cancer cell and the third stretch of consecutive aminoacids of the fusion protein induces a phenotype in the cancer cell or ina non-malignant cell in the subject; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

In some embodiments, the effective amount of the radiation or thechemotherapeutic agent is less than the amount that would be effectiveto treat the subject if the radiation or chemotherapeutic agent wasadministered without the anti-p185her2/neu antibody or the fusionprotein.

In some embodiments, the effective amount of the radiation or thechemotherapeutic agent is less than the amount that would be effectiveto treat the subject if the radiation or chemotherapeutic agent wasadministered without the anti-p185her2/neu antibody and IFNγ or thefusion protein and IFNγ.

In some embodiments, the anti-p185her2/neu antibody or the fusionprotein is administered to the subject in an amount that is less thanthe amount that would be effective to treat the subject if theanti-p185her2/neu antibody or the fusion protein was administeredwithout IFNγ.

In some embodiments, the anti-p185her2/neu antibody inhibits formationof p185her2/neu-containing ErbB protein dimers that produce elevatedtyrosine kinase activity in the cancer cell, thereby inhibitingp185her2/neu signaling in the cancer cell.

In some embodiments, the IFNγ induces the phenotype of class I majorhistocompatibility complex (MHC) antigen expression in the cancer cell.

In some embodiments, the IFNγ induces the phenotype of a reduced abilityto attract an immune suppressor cell to migrate into themicroenvironment of the cancer cell or the non-malignant cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the IFNγ induces the phenotype of class I MHCantigen expression in the non-malignant cell.

In some embodiments, the IFNγ inhibits the malignant transformation ofthe non-malignant cell or increases the differentiation of thenon-malignant cell.

In some embodiments, the IFNγ induces a cytostatic phenotype in thenon-malignant cell.

In some embodiments, the IFNγ induces the phenotype of acceleratedand/or maintained degradation of Snail or Slug in the non-malignantcell.

In some embodiments, the IFNγ induces the phenotype of increasedsensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the IFNγ induces the phenotype of a reduced abilityto evade the immune system of the subject.

In some embodiments, the first stretch of consecutive amino acids of thefusion protein inhibits formation of p185her2/neu-containing ErbBprotein dimers that produce elevated tyrosine kinase activity in thecancer cell, thereby inhibiting p185her2/neu signaling in the cancercell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of class I major histocompatibilitycomplex (MHC) antigen expression in the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of a reduced ability to attract animmune suppressor cell to migrate into the microenvironment of thecancer cell or the non-malignant cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of class I MHC antigen expressionin the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein inhibits the malignant transformation of thenon-malignant cell or increases the differentiation of the non-malignantcell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces a cytostatic phenotype in the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of accelerated and/or maintaineddegradation of Snail or Slug in the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of increased sensitivity toradiation or a chemotherapeutic agent.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of a reduced ability to evade theimmune system of the subject.

In some embodiments, the non-malignant cell is a stem cell-like cell, adedifferentiated cell, and/or a cell that has undergone or is undergoingan epithelial to mesenchymal transition (EMT).

In some embodiments, the non-malignant cell is in a tumor with thecancer cell or is in the microenvironment of the cancer cell.

The present invention provides a method of treating a subject afflictedwith cancer or preventing the development of a tumor in a subject atrisk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an anti-p185her2/neu antibody whichinhibits p185her2/neu signaling in the cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and concurrently or subsequently administering IFNγ whichinduces further phenotypic change in the cancer cell; or

ii) administering to the subject a fusion protein of the invention,wherein the first stretch of consecutive amino acids of the fusionprotein inhibits p185her2/neu signaling in a cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and the third stretch of consecutive amino acids of the fusionprotein induces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

In some embodiments, the anti-p185her2/neu antibody inhibits formationof p185her2/neu-containing ErbB protein dimers that produce elevatedtyrosine kinase activity in the cancer cell, thereby inhibitingp185her2/neu signaling in the cancer cell.

In some embodiments, the anti-p185her2/neu antibody converts thephenotype of the cancer cell to

i) a cytostatic phenotype;

ii) a less malignant phenotype;

iii) a stem cell-like phenotype;

iv) a less dedifferentiated phenotype;

v) a more epithelial phenotype; or

vi) a less mesenchymal phenotype.

In some embodiments, the anti-p185her2/neu antibody or the IFNγ inducesthe phenotype of a reduced ability to attract an immune suppressor cellto migrate into the microenvironment of the cancer cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the anti-p185her2/neu antibody or the IFNγ inducesthe phenotype of class I major histocompatibility complex (MHC) antigenexpression in the cancer cell.

In some embodiments, the anti-p185her2/neu antibody or the IFNγ inducesthe phenotype of accelerated or maintained degradation of Snail or Slugin the cancer cell.

In some embodiments, the anti-p185her2/neu antibody or the IFNγ inducesthe phenotype of

-   -   a) a reduced level of p185her2/neu protein on the surface of the        cancer cell;    -   b) increased KLF4 expression in the cancer cell;    -   c) reduced expression of ALDH1 in the cancer cell;    -   d) increased effector T cell activity against the cancer cell;        or    -   e) increased accumulation of cytolytic anti-tumor M1 macrophages        in the microenvironment of the cancer cell, wherein the cancer        cell is in a tumor.

In some embodiments, the IFNγ induces a cytostatic phenotype in thecancer cell.

In some embodiments, the IFNγ increases the differentiation of thecancer cell.

In some embodiments, the IFNγ induces the further phenotypic change ofincreased sensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the IFNγ induces the further phenotypic change of areduced ability to evade the immune system of the subject.

In some embodiments, the first stretch of consecutive amino acids of thefusion protein inhibits formation of p185her2/neu-containing ErbBprotein dimers that produce elevated tyrosine kinase activity in thecancer cell, thereby inhibiting p185her2/neu signaling in the cancercell.

In some embodiments, the anti-p185her2/neu antibody converts thephenotype of the cancer cell to

i) a cytostatic phenotype;

ii) a less malignant phenotype;

iii) a stem cell-like phenotype;

iv) a less dedifferentiated phenotype;

v) a more epithelial phenotype; or

vi) a less mesenchymal phenotype.

In some embodiments, the anti-p185her2/neu antibody or the third stretchof consecutive amino acids of the fusion protein induces the phenotypeof a reduced ability to attract an immune suppressor cell to migrateinto the microenvironment of the cancer cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the anti-p185her2/neu antibody or the third stretchof consecutive amino acids of the fusion protein induces the phenotypeof class I major histocompatibility complex (MHC) antigen expression inthe cancer cell.

In some embodiments, the anti-p185her2/neu antibody or the third stretchof consecutive amino acids of the fusion protein induces the phenotypeof accelerated or maintained degradation of Snail or Slug in the cancercell.

In some embodiments, the anti-p185her2/neu antibody or the third stretchof consecutive amino acids of the fusion protein induces the phenotypeof

-   -   a) a reduced level of p185her2/neu protein on the surface of the        cancer cell;    -   b) increased KLF4 expression in the cancer cell;    -   c) reduced expression of ALDH1 in the cancer cell;    -   d) increased effector T cell activity against the cancer cell;        or    -   e) increased accumulation of cytolytic anti-tumor M1 macrophages        in the microenvironment of the cancer cell, wherein the cancer        cell is in a tumor.

In some embodiments, the combination of the anti-p185her2/neu antibodyand IFNγ alters the stem cell-ness of the cancer cell. In an embodimentthe combination reduces the stem cell-ness of the cancer cell. In someembodiments, reducing the stem cell-ness of the cancer comprisesincreasing differentiation of the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces a cytostatic phenotype in the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein increases the differentiation of the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the further phenotypic change of increasedsensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the further phenotypic change of a reducedability to evade the immune system of the subject.

In some embodiments, the phenotype of the cancer cell is converted tothe phenotype of a non- or less-malignant cell that is a stem cell-likecell, a dedifferentiated cell, and/or a cell that has undergone or isundergoing an epithelial to mesenchymal transition (EMT).

The present invention provides a method of treating a subject afflictedwith a tumor associated with p185her2/neu or preventing development of atumor associated with p185her2/neu in a subject, which comprisesadministering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of treating a subject afflictedwith a tumor associated with p185her2/neu or preventing development of atumor associated with p185her2/neu in a subject, which comprisesadministering to the subject a composition including

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention,

and a pharmaceutically acceptable carrier.

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress p185her2/neu in a subjectin need of such inhibition which comprises administering to said subject

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressedp185her2/neu and inhibit the development of said breast cells thatoverexpress p185her2/neu into breast cancer cells.

In some embodiments, the anti-p185her2/neu antibody is administered tothe subject before the IFNγ.

In some embodiments, the anti-p185her2/neu antibody is administered tothe subject at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 daysbefore IFNγ is administered to the subject.

In some embodiments,

i) the anti-p185her2/neu antibody and the IFNγ; or

ii) the fusion protein of the invention,

is administered to the subject before the radiation or thechemotherapeutic agent.

In some embodiments,

i) the anti-p185her2/neu antibody and the IFNγ; or

ii) the fusion protein of the invention,

is administered to the subject at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4. 4.5, or 5 days before the radiation or the chemotherapeuticagent is administered to the subject.

In some embodiments, the method comprises administering achemotherapeutic agent to the subject.

In some embodiments, the chemotherapeutic agent is administered to thesubject in an amount that is less than the amount that would beeffective to treat the subject if the chemotherapeutic agent wasadministered without the anti-p185her2/neu antibody and the IFNγ or thefusion protein and the IFNγ.

In some embodiments, the chemotherapeutic agent is a cytotoxic agent.

In some embodiments, the cytotoxic agent is a taxane or a platinum-basedchemotherapeutic agent.

In some embodiments, the method comprises administering radiation to thesubject.

In some embodiments, the radiation is administered to the subject in anamount that is less than the amount that would be effective to treat thesubject if the radiation was administered without the anti-p185her2/neuantibody and the IFNγ or the fusion protein and the IFNγ.

In some embodiments, the radiation is ionizing radiation.

In some embodiments, the ionizing radiation is gamma radiation.

In some embodiments, the cancer is associated with p185her2/neu.

In some embodiments, cells of the cancer have more p185her2/neu activitythan cells from normal tissue of the same type.

In some embodiments, cells of the cancer express p185her2/neu at ahigher level than cells from normal tissue of the same type.

In some embodiments, the cancer is in the form of, or comprises at leastone tumor.

In some embodiments, administering to the subject

i) an anti-p185her2/neu antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention,

is effective to reduce cancer cell proliferation in the tumor and themigration of immune suppressor cells into the tumor.

In some embodiments, the cancer is an adenocarcinoma.

In some embodiments, the cancer is glioblastoma, prostate cancer, lungcancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer,or stomach cancer.

In some embodiments, the cancer is breast cancer, and the breast canceris ductal carcinoma in situ (DCIS).

In some embodiments, the cancer is breast cancer and the breast canceris

-   -   a) estrogen receptor positive;    -   b) estrogen receptor negative;    -   c) Her2 positive;    -   d) Her2 negative;    -   e) progesterone receptor positive;    -   f) progesterone receptor negative; or    -   g) any combination of a) through f).

In some embodiments, treating the subject comprises preventing orreducing tumor growth in the subject.

In some embodiments, treating the subject comprises completely arrestingcancer cell growth in the subject.

In some embodiments, treating the subject comprises increased lysis ofcancer cells in the subject.

In some embodiments, the subject is treated such that an increase in thevolume of the at least one tumor cannot be detected for a period of atleast 30 days during or after treatment.

In some embodiments, the subject is a mammalian subject.

In some embodiments, the mammalian subject is a human subject.

In some embodiments, the anti-p185her2/neu antibody is administeredtwice per week, and the IFNγ is administered three times per week.

In some embodiments, the anti-p185her2/neu antibody is a monoclonalantibody.

In some embodiments, the anti-p185her2/neu antibody is 4D5, pertuzumab,trastuzumab, or 7.16.4.

In some embodiments, the anti-p185her2/neu antibody binds at least aportion of the same epitope as trastuzumab.

In some embodiments, the method further comprises administering a secondantibody to the subject.

In some embodiments, the second antibody is an anti-p185her2/neuantibody.

In some embodiments, two anti-p185her2/neu antibodies are administeredto the subject, and each anti-p185her2/neu antibody targets a differentepitope of p185her2/neu.

In some embodiments, the second antibody is an anti-EGFR antibody.

In some embodiments, the anti-EGFR antibody is cetuximab.

In some embodiments, the second antibody is an anti-PD1 antibody.

In some embodiments, the second antibody is an anti-PD1 antibody or ananti-PD-L1 antibody.

In some embodiments, each of the antibodies is administered to thesubject before IFNγ is administered to the subject.

In some embodiments, each of the antibodies is administered to thesubject at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days beforeIFNγ is administered to the subject.

In some embodiments, IFNγ is administered to the subject concomitantlywith the antibodies, or within 24 hours after the antibodies areadministered to the subject.

In some embodiments, the second antibody is administered to the subjectafter IFNγ is administered to the subject.

In some embodiments, the second antibody is administered to the subjectat least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days after IFNγ isadministered to the subject.

In some embodiments, the second antibody is a monoclonal antibodies.

In some embodiments, the anti-PD1 antibody or anti-PD-L1 antibody isadministered after the anti-p185her2/neu antibody and IFNγ.

In some embodiments, the anti-p185her2/neu antibody or the fusionprotein is administered to the subject in an amount that is less thanthe amount that would be effective to treat the subject if theanti-p185her2/neu antibody or the fusion protein was administeredwithout IFNγ.

The present invention provides a composition for the treatment of asubject afflicted with cancer, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing cancer toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing a tumor toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for preventing thedevelopment of a tumor in a subject at risk of developing the tumor,comprising

i) the fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a combination for the treatment of asubject afflicted with cancer or preventing the development of a tumorin a subject at risk of developing the tumor, comprising

i) the fusion protein of the invention or ii) an anti-p185her2/neuantibody and interferon-gamma (IFNγ), and

ii) a second antibody,

and a pharmaceutically acceptable carrier.

The present invention provides a polynucleotide encoding the fusionprotein of the invention.

The present invention provides an expression vector comprising apolynucleotide of the invention operably linked to a promoter.

The present invention provides a cell comprising a expression vector ofthe invention.

The present invention provides a fusion protein comprising

(a) a first stretch of consecutive amino acids, the sequence of which isthe sequence of an anti-EGFR polypeptide;

(b) a second stretch of consecutive amino acids, the sequence of whichis the sequence of a polypeptide capable of binding at least onepolypeptide other than EGFR; and

(c) a third stretch of consecutive amino acids, the sequence of whichcomprises the sequence of a biologically active portion ofinterferon-gamma (IFNγ),

wherein (b) is located at the carboxy-terminal end of (a), and (c) islocated at the carboxy-terminal end of (b).

In some embodiments, the fusion protein further comprises anoligopeptide linker between (a) and (b).

In some embodiments, the fusion protein further comprises anoligopeptide linker between (b) and (c).

In some embodiments, the fusion protein further comprises a secondoligopeptide linker between (b) and (c).

In some embodiments, the amino acid sequence of the oligopeptide linkeris identical to the amino acid sequence of the second oligopeptidelinker.

In some embodiments, the amino acid sequence of the oligopeptide linkeris different from the amino acid sequence of the second oligopeptidelinker.

In some embodiments,

i) the oligopeptide is a polyglycine oligopeptide linker or aglycine-serine oligopeptide linker; and

ii) the second oligopeptide is independently a polyglycine oligopeptidelinker or a glycine-serine oligopeptide linker.

In some embodiments, the amino acid sequence of the polyglycineoligopeptide linker comprises at least two, three, four, five, six,seven, eight, nine, or ten, consecutive glycine residues.

In some embodiments, the amino acid sequence of the glycine-serineoligopeptide linker comprises at least two, three, four, five, six,seven, eight, nine, or ten, consecutive glycine residues.

In some embodiments, the C-terminal residue of the glycine-serineoligopeptide linker is a serine residue.

In some embodiments, (a) is directly contiguous with (b).

In some embodiments, (b) is directly contiguous with (c).

In some embodiments, the anti-EGFR polypeptide is a chain of an antibodyor a portion thereof.

In some embodiments, the antibody chain is a single chain variablefragment (scFv).

In some embodiments, the antibody chain is a monoclonal antibody chain.

In some embodiments, the monoclonal antibody chain is a human monoclonalantibody chain, a humanized monoclonal antibody chain, or a chimericantibody chain.

In some embodiments, the monoclonal antibody chain is a chimericantibody chain, and wherein a portion of the chimeric antibody chain isderived from a human antibody chain.

In some embodiments, the antibody is an anti-EGFR antibody.

In some embodiments, the anti-EGFR antibody is a monoclonal antibody.

In some embodiments, the anti-EGFR monoclonal antibody is a humanmonoclonal antibody.

In some embodiments, the anti-EGFR monoclonal antibody is a humanizedmonoclonal antibody.

In some embodiments, the anti-EGFR antibody binds at least a portion ofthe same epitope as cetuximab.

In some embodiments, the anti-EGFR antibody is cetuximab.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than EGFR is capable of binding at least one antibody.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than EGFR is capable of binding at least one antibodyFc-region.

In some embodiments, the polypeptide that is capable of binding apolypeptide other than EGFR is derived from a portion of Protein A orProtein G that is capable of binding at least one antibody Fc-region.

In some embodiments, the least one antibody is endogenously expressed ina mammal.

In some embodiments, the sequence of the third stretch of consecutiveamino acids is identical to the sequence of IFNγ.

In some embodiments, the sequence of the fusion protein has beenmodified to reduce immunogenicity in a human.

In some embodiments, (a), (b), or (c) has been humanized.

In some embodiments, each of (a), (b), and (c) has been humanized.

In some embodiments, the sequence of each of (a), (b), and (c) is foundin an endogenous human polypeptide.

In some embodiments, the fusion protein is 4D5scFvZZ-IFNγ, or ahumanized derivative thereof.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises administering to the subject atherapeutically effective amount of the fusion protein of the invention.

In some embodiments, the method further comprises administering anantibody to the subject.

In some embodiments, the antibody is an anti-p185her2/neu antibody.

In some embodiments, the antibody is an anti-EGFR antibody.

In some embodiments, the anti-EGFR antibody is cetuximab.

In some embodiments, the antibody is an anti-PD1 or an anti-PD-L1antibody.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the method further comprises administering achemotherapeutic agent to the subject.

In some embodiments, the chemotherapeutic agent is administered to thesubject in an amount that is less than the amount that would beeffective to treat the subject if the chemotherapeutic agent wasadministered without the fusion protein.

In some embodiments, the method further comprises administering aradiation to the subject.

In some embodiments, the radiation is administered to the subject in anamount that is less than the amount that would be effective to treat thesubject if the radiation was administered without the fusion protein.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of sensitizing cells of a cancerin a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention, and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention provides a method of treating a subject afflictedwith cancer, which comprises

a) sensitizing the cancer to radiation or a chemotherapeutic agent byadministering to the subject

i) an anti-EGFR antibody which inhibits EGFR signaling in a cancer cell,wherein said inhibition induces a phenotype in the cancer cell, andinterferon-gamma (IFNγ) which induces a further phenotype in the cancercell or in a non-malignant cell in the subject; or

ii) a fusion protein of the invention, wherein the first stretch ofconsecutive amino acids of the fusion protein inhibits EGFR signaling ina cancer cell, wherein said inhibition induces a cytostatic phenotype inthe cancer cell and the third stretch of consecutive amino acids of thefusion protein induces a phenotype in the cancer cell or in anon-malignant cell in the subject; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

In some embodiments, the effective amount of the radiation or thechemotherapeutic agent is less than the amount that would be effectiveto treat the subject if the radiation or chemotherapeutic agent wasadministered without the anti-EGFR antibody or the fusion protein.

In some embodiments, the effective amount of the radiation or thechemotherapeutic agent is less than the amount that would be effectiveto treat the subject if the radiation or chemotherapeutic agent wasadministered without the anti-EGFR antibody and IFNγ or the fusionprotein and IFNγ.

In some embodiments, the anti-EGFR antibody or the fusion protein isadministered to the subject in an amount that is less than the amountthat would be effective to treat the subject if the anti-EGFR antibodyor the fusion protein was administered without IFNγ.

In some embodiments, the anti-EGFR antibody inhibits formation ofEGFR-containing ErbB protein dimers that produce elevated tyrosinekinase activity in the cancer cell, thereby inhibiting EGFR signaling inthe cancer cell.

In some embodiments, the IFNγ induces the phenotype of class I majorhistocompatibility complex (MHC) antigen expression in the cancer cell.

In some embodiments, the IFNγ induces the phenotype of a reduced abilityto attract an immune suppressor cell to migrate into themicroenvironment of the cancer cell or the non-malignant cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the IFNγ induces the phenotype of class I MHCantigen expression in the non-malignant cell.

In some embodiments, the IFNγ inhibits the malignant transformation ofthe non-malignant cell or increases the differentiation of thenon-malignant cell.

In some embodiments, the IFNγ induces a cytostatic phenotype in thenon-malignant cell.

In some embodiments, the IFNγ induces the phenotype of acceleratedand/or maintained degradation of Snail or Slug in the non-malignantcell.

In some embodiments, the IFNγ induces the phenotype of increasedsensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the IFNγ induces the phenotype of a reduced abilityto evade the immune system of the subject.

In some embodiments, the first stretch of consecutive amino acids of thefusion protein inhibits formation of EGFR-containing ErbB protein dimersthat produce elevated tyrosine kinase activity in the cancer cell,thereby inhibiting EGFR signaling in the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of class I major histocompatibilitycomplex (MHC) antigen expression in the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of a reduced ability to attract animmune suppressor cell to migrate into the microenvironment of thecancer cell or the non-malignant cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of class I MHC antigen expressionin the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein inhibits the malignant transformation of thenon-malignant cell or increases the differentiation of the non-malignantcell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces a cytostatic phenotype in the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of accelerated and/or maintaineddegradation of Snail or Slug in the non-malignant cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of increased sensitivity toradiation or a chemotherapeutic agent.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the phenotype of a reduced ability to evade theimmune system of the subject.

In some embodiments, the non-malignant cell is a stem cell-like cell, adedifferentiated cell, and/or a cell that has undergone or is undergoingan epithelial to mesenchymal transition (EMT).

In some embodiments, the non-malignant cell is in a tumor with thecancer cell or is in the microenvironment of the cancer cell.

The present invention provides a method of treating a subject afflictedwith cancer or preventing the development of a tumor in a subject atrisk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an anti-EGFR antibody which inhibitsEGFR signaling in the cancer cell, wherein said inhibition converts thephenotype of the cancer cell such that the cancer cell is amenable tofurther phenotypic change by interferon-gamma (IFNγ), and concurrentlyor subsequently administering IFNγ which induces further phenotypicchange in the cancer cell; or

ii) administering to the subject a fusion protein of the invention,wherein the first stretch of consecutive amino acids of the fusionprotein inhibits EGFR signaling in a cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and the third stretch of consecutive amino acids of the fusionprotein induces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

In some embodiments, the anti-EGFR antibody inhibits formation ofEGFR-containing ErbB protein dimers that produce elevated tyrosinekinase activity in the cancer cell, thereby inhibiting EGFR signaling inthe cancer cell.

In some embodiments, the anti-EGFR antibody converts the phenotype ofthe cancer cell to

i) a cytostatic phenotype;

ii) a less malignant phenotype;

iii) a stem cell-like phenotype;

iv) a less dedifferentiated phenotype;

v) a more epithelial phenotype; or

vi) a less mesenchymal phenotype.

In some embodiments, the anti-EGFR antibody or the IFNγ induces thephenotype of a reduced ability to attract an immune suppressor cell tomigrate into the microenvironment of the cancer cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the anti-EGFR antibody or the IFNγ induces thephenotype of class I major histocompatibility complex (MHC) antigenexpression in the cancer cell.

In some embodiments, the anti-EGFR antibody or the IFNγ induces thephenotype of accelerated or maintained degradation of Snail or Slug inthe cancer cell.

In some embodiments, the anti-EGFR antibody or the IFNγ induces thephenotype of

-   -   a) a reduced level of p185her2/neu protein on the surface of the        cancer cell;    -   b) increased KLF4 expression in the cancer cell;    -   c) reduced expression of ALDH1 in the cancer cell;    -   d) increased effector T cell activity against the cancer cell;        or    -   e) increased accumulation of cytolytic anti-tumor M1 macrophages        in the microenvironment of the cancer cell, wherein the cancer        cell is in a tumor.

In some embodiments, the IFNγ induces a cytostatic phenotype in thecancer cell.

In some embodiments, the IFNγ increases the differentiation of thecancer cell.

In some embodiments, the IFNγ induces the further phenotypic change ofincreased sensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the IFNγ induces the further phenotypic change of areduced ability to evade the immune system of the subject.

In some embodiments, the first stretch of consecutive amino acids of thefusion protein inhibits formation of EGFR-containing ErbB protein dimersthat produce elevated tyrosine kinase activity in the cancer cell,thereby inhibiting EGFR signaling in the cancer cell.

In some embodiments, the anti-EGFR antibody converts the phenotype ofthe cancer cell to

i) a cytostatic phenotype;

ii) a less malignant phenotype;

iii) a stem cell-like phenotype;

iv) a less dedifferentiated phenotype;

v) a more epithelial phenotype; or

vi) a less mesenchymal phenotype.

In some embodiments, the anti-EGFR antibody or the third stretch ofconsecutive amino acids of the fusion protein induces the phenotype of areduced ability to attract an immune suppressor cell to migrate into themicroenvironment of the cancer cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the anti-EGFR antibody or the third stretch ofconsecutive amino acids of the fusion protein induces the phenotype ofclass I major histocompatibility complex (MHC) antigen expression in thecancer cell.

In some embodiments, the anti-EGFR antibody or the third stretch ofconsecutive amino acids of the fusion protein induces the phenotype ofaccelerated or maintained degradation of Snail or Slug in the cancercell.

In some embodiments, the anti-EGFR antibody or the third stretch ofconsecutive amino acids of the fusion protein induces the phenotype of

-   -   a) a reduced level of p185her2/neu protein on the surface of the        cancer cell;    -   b) increased KLF4 expression in the cancer cell;    -   c) reduced expression of ALDH1 in the cancer cell;    -   d) increased effector T cell activity against the cancer cell;        or    -   e) increased accumulation of cytolytic anti-tumor M1 macrophages        in the microenvironment of the cancer cell, wherein the cancer        cell is in a tumor.

In some embodiments, the combination of the anti-EGFR antibody and IFNγalters the stem cell-ness of the cancer cell. In an embodiment thecombination reduces the stem cell-ness of the cancer cell. In someembodiments, reducing the stem cell-ness of the cancer comprisesincreasing differentiation of the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces a cytostatic phenotype in the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein increases the differentiation of the cancer cell.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the further phenotypic change of increasedsensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the third stretch of consecutive amino acids of thefusion protein induces the further phenotypic change of a reducedability to evade the immune system of the subject.

In some embodiments, the phenotype of the cancer cell is converted tothe phenotype of a non- or less-malignant cell that is a stem cell-likecell, a dedifferentiated cell, and/or a cell that has undergone or isundergoing an epithelial to mesenchymal transition (EMT).

The present invention provides a method of treating a subject afflictedwith a tumor associated with EGFR or preventing development of a tumorassociated with EGFR in a subject, which comprises administering to thesubject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention.

The present invention also provides a method of treating a subjectafflicted with a tumor associated with EGFR or preventing development ofa tumor associated with EGFR in a subject, which comprises administeringto the subject a composition including

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention,

and a pharmaceutically acceptable carrier.

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress EGFR in a subject in needof such inhibition which comprises administering to said subject

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressed p185 andinhibit the development of said breast cells that overexpress p185 intobreast cancer cells.

In some embodiments, the anti-EGFR antibody is administered to thesubject before the IFNγ.

In some embodiments, the anti-EGFR antibody is administered to thesubject at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days beforeIFNγ is administered to the subject.

In some embodiments,

i) the anti-EGFR antibody and the IFNγ; or

ii) the fusion protein of the invention,

is administered to the subject before the radiation or thechemotherapeutic agent.

In some embodiments,

i) the anti-EGFR antibody and the IFNγ; or

ii) the fusion protein of the invention,

is administered to the subject at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4. 4.5, or 5 days before the radiation or the chemotherapeuticagent is administered to the subject.

In some embodiments, the method further comprises administering achemotherapeutic agent to the subject.

In some embodiments, the chemotherapeutic agent is administered to thesubject in an amount that is less than the amount that would beeffective to treat the subject if the chemotherapeutic agent wasadministered without the anti-EGFR antibody and the IFNγ or the fusionprotein and the IFNγ.

In some embodiments, the chemotherapeutic agent is a cytotoxic agent.

In some embodiments, the cytotoxic agent is a taxane or a platinum-basedchemotherapeutic agent.

In some embodiments, the method further comprises administeringradiation to the subject.

In some embodiments, the radiation is administered to the subject in anamount that is less than the amount that would be effective to treat thesubject if the radiation was administered without the anti-p185her2/neuantibody and the IFNγ or the fusion protein and the IFNγ.

In some embodiments, the radiation is ionizing radiation.

In some embodiments, the ionizing radiation is gamma radiation.

In some embodiments, the cancer is associated with EGFR.

In some embodiments, cells of the cancer have more EGFR activity thancells from normal tissue of the same type.

In some embodiments, cells of the cancer express EGFR at a higher levelthan cells from normal tissue of the same type.

In some embodiments, the cancer is in the form of, or comprises at leastone tumor.

In some embodiments, administering to the subject

i) an anti-EGFR antibody and interferon-gamma (IFNγ); or

ii) a fusion protein of the invention,

is effective to reduce cancer cell proliferation in the tumor and themigration of immune suppressor cells into the tumor.

In some embodiments, the cancer is an adenocarcinoma.

In some embodiments, the cancer is glioblastoma, prostate cancer, lungcancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer,or stomach cancer.

In some embodiments, the cancer is breast cancer, and the beast canceris DCIS.

In some embodiments, the cancer is breast cancer and the breast canceris

-   -   a) estrogen receptor positive;    -   b) estrogen receptor negative;    -   c) Her2 positive;    -   d) Her2 negative;    -   e) progesterone receptor positive;    -   f) progesterone receptor negative; or    -   g) any combination of a) through f).

In some embodiments, treating the subject comprises preventing orreducing tumor growth.

In some embodiments, the subject is treated such that an increase in thevolume of the at least one tumor cannot be detected for a period of atleast 30 days during or after treatment.

In some embodiments, treating the subject comprises completely arrestingcancer cell growth in the subject.

In some embodiments, treating the subject comprises increased lysis ofcancer cells in the subject.

In some embodiments, the subject is a mammalian subject.

In some embodiments, the mammalian subject is a human subject.

In some embodiments, the anti-EGFR antibody is administered twice perweek, and the IFNγ is administered three times per week.

In some embodiments, the anti-EGFR antibody is a monoclonal antibody.

In some embodiments, the anti-EGFR antibody is cetuximab.

In some embodiments, the anti-EGFR antibody binds at least a portion ofthe same epitope as cetuximab.

In some embodiments, the method further comprises administering a secondantibody to the subject.

In some embodiments, the second antibody is an anti-EGFR antibody.

In some embodiments, the second antibody is an anti-PD1 antibody or ananti-PD-L1 antibody.

In some embodiments, the second antibody is an anti-p185her2/neuantibody.

In some embodiments, the anti-p185her2/neu antibody is trastuzumab.

In some embodiments, each of the antibodies is administered to thesubject before IFNγ is administered to the subject.

In some embodiments, each of the antibodies is administered to thesubject at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days beforeIFNγ is administered to the subject.

In some embodiments, IFNγ is administered to the subject concomitantlywith the antibodies, or within 24 hours after the antibodies areadministered to the subject.

In some embodiments, the second antibody is administered to the subjectafter IFNγ is administered to the subject.

In some embodiments, the second antibody is administered to the subjectat least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days after IFNγ isadministered to the subject.

In some embodiments, the second antibody is a monoclonal antibodies.

In some embodiments, the anti-PD1 antibody or anti-PD-L1 antibody isadministered after the anti-EGFR antibody and IFNγ.

In some embodiments, the anti-EGFR antibody or the fusion protein isadministered to the subject in an amount that is less than the amountthat would be effective to treat the subject if the anti-EGFR antibodyor the fusion protein was administered without IFNγ.

The present invention provides a composition for the treatment of asubject afflicted with cancer, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing cancer toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for sensitizing a tumor toradiation or a chemotherapeutic agent, comprising

i) a fusion protein of the invention; or

ii) an anti-EGFR antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a composition for preventing thedevelopment of a tumor in a subject at risk of developing the tumor,comprising

i) the fusion protein of the invention; or

ii) an anti-p185her2/neu antibody and interferon-gamma (IFNγ),

and a pharmaceutically acceptable carrier.

The present invention provides a combination for the treatment of asubject afflicted with cancer or preventing the development of a tumorin a subject at risk of developing the tumor, comprising

i) the fusion protein of the invention or ii) an anti-p185her2/neuantibody and interferon-gamma (IFNγ), and

ii) a second antibody,

and a pharmaceutically acceptable carrier.

The present invention provides a polynucleotide encoding a fusionprotein of the invention.

The present invention provides an expression vector comprising apolynucleotide of the invention operably linked to a promoter.

The present invention provides a cell comprising a expression vector ofthe invention.

In some embodiments, the method further comprises administering to thesubject an EGFr inhibitor.

In some embodiments, the EGFRr inhibitor inhibits the kinase activity ofp185her2/neu or EGFR.

In some embodiments, the EGFRr inhibitor is C318, gefitinib, erlotinib,lapatinib, or vandetanib, or a pharmaceutically acceptable salt or esterthereof.

In some embodiments, the EGFRr inhibitor is an organic compound having amolecular weight less than 1000 Daltons.

In some embodiments of the invention relating to an anti-p185her2/neupolypeptide (AHNP), the AHNP has the amino acid sequence set forth inSEQ ID NO: 3.

The present invention provides a method of treating a subject afflictedwith a tumor or preventing development of a tumor in a subject, whichcomprises administering to the subject

i) at least one antibody; and

ii) interferon-gamma (IFNγ).

In some embodiments, the at least one antibody is a monoclonal antibody.

In some embodiments, the at least one antibody is one, two, three, four,five or more antibodies, comprising at least one an anti-p185her2/neuantibody, at least one anti-EGFR antibody, at least one anti-PD1antibody, or at least one anti-PD-L1 antibody.

In some embodiments, the at least one antibody is administered beforeIFNγ.

The present invention provides a fusion protein comprising

(i) a first stretch of consecutive amino acids, the sequence of which isthe sequence of an anti-p185her2/neu polypeptide or an anti-EGFRpolypeptide; and

(ii) a second stretch of consecutive amino acids, the sequence of whichcomprises the sequence of a biologically active portion ofinterferon-gamma (IFNγ),

wherein (ii) is located at the carboxy-terminal end of (i).

In some embodiments, the fusion protein further comprises anoligopeptide linker between (i) and (ii).

In some embodiments, a linker within a fusion protein of the inventioncomprises a labile cleavage site.

In some embodiments, the anti-p185her2/neu polypeptide or the anti-EGFRpolypeptide is a chain of an antibody or a portion thereof.

In some embodiments, the antibody chain is a single chain variablefragment (scFv).

In some embodiments, the antibody chain is a monoclonal antibody chain.

In some embodiments, the monoclonal antibody chain is a human monoclonalantibody chain, a humanized monoclonal antibody chain, or a chimericantibody chain.

In some embodiments, wherein the monoclonal antibody chain is a chimericantibody chain, and wherein a portion of the chimeric antibody chain isderived from a human antibody chain.

In some embodiments, the antibody is an anti-p185her2/neu antibody or ananti-EGFR antibody.

In some embodiments, the fusion protein further comprises (iii) a thirdstretch of consecutive amino acids, the sequence of which comprises thesequence of a biologically active portion of interferon-gamma (IFNγ). Insome embodiments, (iii) is located at the carboxy-terminal end of (ii)

In some embodiments, the fusion protein further comprises anoligopeptide linker between (ii) and (iii).

In some embodiments, the fusion protein further comprises the linkercomprises a labile cleavage site.

In some embodiments, the fusion protein further comprises the sequenceof the second or the third stretch of consecutive amino acids isidentical to the sequence of IFNγ.

In some embodiments, the fusion protein comprises an IFNγ dimer.

In some embodiments, a biologically active portion of IFNγ is a portionof IFNγ that is biologically active. In some embodiments, a biologicallyactive portion of IFNγ is full-length IFNγ.

The present invention provides a method of sensitizing cancer cells toradiation or a chemotherapeutic agent, which comprises contacting thecancer cells with

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

The present invention also provides a method of sensitizing cells of acancer in a subject afflicted with the cancer to radiation or achemotherapeutic agent, which comprises administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

The present invention further provides a method of treating a subjectafflicted with cancer, which comprises

a) sensitizing cells of the cancer, and cells that have undergoneepithelial to mesenchymal transition (EMT) or are undergoing EMT, toradiation or a chemotherapeutic agent by administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ), and

b) thereafter administering radiation or a chemotherapeutic agent to thesubject.

The present invention also provides a method of treating a subjectafflicted with cancer or preventing the development of a tumor in asubject at risk of developing the tumor, which comprises

a) sensitizing a cancer cell to radiation or a chemotherapeutic agent by

i) administering to the subject an erbB inhibitor which inhibits EGFRsignaling or p185her2/neu signaling in the cancer cell, wherein saidinhibition converts the phenotype of the cancer cell such that thecancer cell is amenable to further phenotypic change by interferon-gamma(IFNγ), and concurrently or subsequently administering IFNγ whichinduces further phenotypic change in the cancer cell; and

b) thereafter administering a therapeutically effective amount ofradiation or a chemotherapeutic agent to the subject.

In some embodiments, the effective amount of the radiation or thechemotherapeutic agent is less than the amount that would be effectiveto treat the subject if the radiation or chemotherapeutic agent wasadministered without the erbB inhibitor and IFNγ.

In some embodiments, the erbB inhibitor is administered to the subjectin an amount that is less than the amount that would be effective totreat the subject if the erbB inhibitor was administered without IFNγ.

In some embodiments, the erbB inhibitor converts the phenotype of thecancer cell to

i) a cytostatic phenotype;

ii) a less malignant phenotype;

iii) a stem cell-like phenotype;

iv) a less dedifferentiated phenotype;

v) a more epithelial phenotype; or

vi) a less mesenchymal phenotype.

In some embodiments, the erbB inhibitor or the IFNγ induces thephenotype of a reduced ability to attract an immune suppressor cell tomigrate into the microenvironment of the cancer cell.

In some embodiments, the immune suppressor cell is a myeloid-derivedsuppressor cell (MDSC).

In some embodiments, the erbB inhibitor or the IFNγ induces thephenotype of increased class I major histocompatibility complex (MHC)antigen expression in the cancer cell.

In some embodiments, the erbB inhibitor or the IFNγ induces thephenotype of accelerated or maintained degradation of Snail or Slug inthe cancer cell.

In some embodiments, the erbB inhibitor or the IFNγ induces thephenotype of

-   -   a) a reduced level of p185her2/neu protein on the surface of the        cancer cell;    -   b) increased KLF4 expression in the cancer cell;    -   c) reduced expression of ALDH1 in the cancer cell;    -   d) increased effector T cell activity against the cancer cell;        or    -   e) increased accumulation of cytolytic anti-tumor M1 macrophages        in the microenvironment of the cancer cell, wherein the cancer        cell is in a tumor.

In some embodiments, the IFNγ induces a cytostatic phenotype in thecancer cell.

In some embodiments, the IFNγ increases the differentiation of thecancer cell.

In some embodiments, the IFNγ induces the further phenotypic change ofincreased sensitivity to radiation or a chemotherapeutic agent.

In some embodiments, the IFNγ induces the further phenotypic change of areduced ability of the cancer to evade the immune system of the subject.

In some embodiments, the phenotype of the cancer cell is converted tothe phenotype of a non- or less-malignant cell that is a stem cell-likecell, a dedifferentiated cell, or a cell that has undergone or isundergoing an epithelial to mesenchymal transition (EMT).

The present invention provides a method of treating a subject afflictedwith a tumor associated with EGFR or p185her2/neu or preventingdevelopment of a tumor associated with EGFR or p185her2/neu in asubject, which comprises administering to the subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ).

In some embodiments, the erbB inhibitor is administered to the subjectbefore the IFNγ.

In some embodiments, the erbB inhibitor is administered to the subjectat least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 days before IFNγ isadministered to the subject.

In some embodiments, the erbB inhibitor and the IFNγ are administered tothe subject before the radiation or the chemotherapeutic agent.

In some embodiments, the erbB inhibitor and the IFNγ are administered tothe subject at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 daysbefore the radiation or the chemotherapeutic agent is administered tothe subject.

In some embodiments, the method comprises administering achemotherapeutic agent to the subject.

In some embodiments, the chemotherapeutic agent is administered to thesubject in an amount that is less than the amount that would beeffective to treat the subject if the chemotherapeutic agent wasadministered without the erbB inhibitor and the IFNγ.

In some embodiments, the chemotherapeutic agent is a cytotoxic agent.

In some embodiments, the cytotoxic agent is a taxane or a platinum-basedchemotherapeutic agent.

In some embodiments, the method comprises administering radiation to thesubject.

In some embodiments, the radiation is administered to the subject in anamount that is less than the amount that would be effective to treat thesubject if the radiation was administered without the erbB inhibitor andthe IFNγ.

In some embodiments, the radiation is ionizing radiation.

In some embodiments, the ionizing radiation is gamma radiation.

In some embodiments, the cancer is associated with p185her2/neu.

In some embodiments, cells of the cancer have more p185her2/neu activitythan cells from normal tissue of the same type.

In some embodiments, cells of the cancer express p185her2/neu at ahigher level than cells from normal tissue of the same type.

In some embodiments, the cancer is in the form of, or comprises at leastone tumor.

In some embodiments, administering to the subject the erbB inhibitor andthe IFNγ is effective to reduce cancer cell proliferation in the tumoror the migration of immune suppressor cells into the tumor.

In some embodiments, the cancer is an adenocarcinoma.

In some embodiments, the cancer is glioblastoma, prostate cancer, lungcancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer,or stomach cancer.

In some embodiments, the cancer is breast cancer, and the breast canceris ductal carcinoma in situ (DCIS).

In some embodiments, the cancer is breast cancer and the breast canceris

-   -   a) estrogen receptor positive;    -   b) estrogen receptor negative;    -   c) Her2 positive;    -   d) Her2 negative;    -   e) progesterone receptor positive;    -   f) progesterone receptor negative; or    -   g) any combination of a) through f).

In some embodiments, treating the subject comprises preventing orreducing tumor growth in the subject.

In some embodiments, treating the subject comprises completely arrestingcancer cell growth in the subject.

In some embodiments, treating the subject comprises increased lysis ofcancer cells in the subject.

In some embodiments, the subject is treated such that an increase in thevolume of the at least one tumor cannot be detected for a period of atleast 30 days during or after treatment.

In some embodiments, the subject is a mammalian subject.

In some embodiments, the mammalian subject is a human subject.

In some embodiments, the method further comprises administering anantibody to the subject.

In some embodiments, the antibody is an anti-p185her2/neu antibody.

In some embodiments, the antibody is an anti-EGFR antibody.

In some embodiments, the antibody is an anti-PD1 or anti-PD-L1 antibody.

In some embodiments, the erbB inhibitor and the antibody areadministered to the subject before IFNγ is administered to the subject.

In some embodiments, the erbB inhibitor and the antibody areadministered to the subject at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4.4.5, or 5 days before IFNγ is administered to the subject.

In some embodiments, IFNγ is administered to the subject concomitantlywith the erbB inhibitor and the antibody, or within 24 hours after theerbB inhibitor and the antibody are administered to the subject.

In some embodiments, the antibody is administered to the subject afterIFNγ is administered to the subject.

In some embodiments, the antibody is administered to the subject atleast 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4. 4.5, or 5 days after IFNγ isadministered to the subject.

In some embodiments, the antibody is a monoclonal antibody.

The present invention provides a method of inhibiting development intocancer cells of breast cells that overexpress p185her2/neu in a subjectin need of such inhibition which comprises administering to said subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressedp185her2/neu and inhibit the development of said breast cells thatoverexpress p185her2/neu into breast cancer cells.

The present invention also provides a method of inhibiting developmentinto cancer cells of breast cells that overexpress EGFR in a subject inneed of such inhibition which comprises administering to said subject

i) an erbB inhibitor; and

ii) interferon-gamma (IFNγ),

each in a sufficient amount to down regulate the overexpressed p185 andinhibit the development of said breast cells that overexpress p185 intobreast cancer cells.

In some embodiments, the erbB inhibitor is a compound that

a) is in a clinical trial; or

b) is approved for use in human subjects.

In some embodiments, the erbB inhibitor is a p185her2/neu kinaseinhibitor.

In some embodiments, the erbB inhibitor is an EGFR kinase inhibitor.

In some embodiments, the erbB inhibitor is an organic compound having amolecular weight less than 1000 Daltons.

In some embodiments, the erbB inhibitor is gefitinib, erlotinib,lapatinib, or vandetanib, or a pharmaceutically acceptable salt or esterthereof.

Gefitinib, is commercially available from AstraZeneca AB (S-151 85Sodertalje Sweden). The CAS Registry number for gefitinib is184475-35-2. Gefitinib is also known as Iressa. The structure forgefitinib is:

Gefitinib is described in Lynch, et al., “Activating mutations in theepidermal growth factor receptor underlying responsiveness ofnon-small-cell lung cancer to gefitinib,” N. Engl. J. Med., 350: 2129-39(2004); Paez, et al., “EGFR mutations in lung cancer: correlation withclinical response to gefitinib therapy,” Science, 304: 1497-1500 (2004);and U.S. Pat. No. 8,350,029, issued Jan. 8, 2013, the entire contents ofeach of which are hereby incorporated herein in their entireties.

Erlotinib, is commercially available from OSI Pharmaceuticals, LLC(Northbrook, Ill., 60062, USA). Erlotinib is also known as Tarceva. Thestructure for erlotinib is:

Erlotinib is described in U.S. Pat. No. 8,642,758, issued Feb. 4, 2014the entire content of which is hereby incorporated herein in itsentirety.

Lapatinib, is commercially available from GlaxoSmithKline (ResearchTriangle Park, N.C. 27709, USA). Lapatinib is also known as Tykerb. Thestructure for lapatinib is:

Lapatinib is described in Burris H A (2004). “Dual kinase inhibition inthe treatment of breast cancer: initial experience with the EGFR/ErbB-2inhibitor lapatinib”. Oncologist. 9 Suppl 3: 10-5; and U.S. Pat. No.8,664,389, issued Mar. 4, 2014 the entire contents of each of which arehereby incorporated herein in their entireties.

Vandetanib is commercially available from AstraZeneca Pharmaceuticals LP(Wilmington, Del. 19850, USA). The structure of vandetanib is:

Vandetanib is also known as Caprelsa. Vandetanib is described in Martin,P.; Oliver, S.; Kennedy, S. J.; Partridge, E.; Hutchison, M.; Clarke,D.; Giles, P. (2012). “Pharmacokinetics of Vandetanib: Three Phase IStudies in Healthy Subjects”. Clinical Therapeutics 34 (1): 221-237; andU.S. Pat. No. 8,609,673, issued Dec. 17, 2013 the entire contents ofeach of which are hereby incorporated herein in their entireties.

In some embodiments, the erbB inhibitor has the structure:

Compounds of these structures are described in U.S. Patent ApplicationPublication No. US 2014-0135298, published May 15, 2014, the entirecontent of which is incorporated herein by reference.

In some embodiments, the erbB inhibitor has the structure:

-   -   wherein    -   R₁ is independently H, optionally substituted amino, optionally        substituted C₁₋₆ alkyl, optionally substituted C1-6 alkoxy,        optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆        alkynyl, optionally substituted benzyloxy, cyano, halo, hydroxy,        nitro, optionally substituted phenoxy, or mono-, di-, or        trifluoromethyl;    -   n is 1, 2, or 3;    -   R₂ is independently H or C₁₋₃-alkyl;    -   R_(3A) is —OR_(5A), —NR₂R_(5A), —SR_(5A), —C(O)R_(5A),        —C(O)OR_(5A), —C(O)N(R₂)(R_(5A)), —OC(O)R_(5A), —OC(O)OR_(5A),        —OC(O)NR₂R_(5A), —NR₂C(O)R_(5A), —NR₂C(O)OR_(5A);    -   R₄ is H, —N(R₂)₂, optionally substituted C₁₋₃-alkyl, optionally        substituted C₁₋₃-alkoxy, cyano, halo, hydroxy, nitro, or mono-,        di-, or trifluoromethyl;    -   R_(5A) is —(C₁₋₄-alkyl)-X—R₆-R₇;    -   X is independently O, S, or N(R₂);    -   R₆ is a bond or C₅₋₆ aryl or C₅₋₆ heteroaryl;    -   R₇ is either a C₁₋₄-alkyl substituted by at least one —OH or        —C(O)OR₂ or —C(O)N(R₂)₂, or a C₅ heteroaryl containing 1-3        heteroatoms and substituted by R₂ and either a halo-substituted        benzyloxy or —X—R₈; and    -   R₈ is C₁₋₃ alkyl substituted by at least one —OH, —COOH,        —C(O)O—C₁₋₄ alkyl, —C(O)N(R₂)₂, or C₁₋₅ cycloalkyl;    -   or a pharmaceutically acceptable salt form thereof.

Compounds of this structure, as well as processes of synthesizingcompounds of this structure are described in U.S. Patent ApplicationPublication No. US 2014-0309246, published Oct. 16, 2014, the entirecontent of which is incorporated herein by reference.

In some embodiments, the erbB inhibitor has the structure:

-   -   wherein

R₁ is independently H, optionally substituted amino, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionallysubstituted C₁₋₆ alkenyl, optionally substituted C₁₋₆ alkynyl,optionally substituted benzyloxy, cyano, halo, hydroxy, nitro,optionally substituted phenoxy, or mono-, di-, or trifluoromethyl;

n is 1, 2, or 3;

R₂ is independently H or C1_3-alkyl;

R_(3B) is —OR_(5B), —NR₂R_(5B), —SR_(5B), —C(O)R_(5B), —C(O)OR_(5B),—C(O)N(R₂)(R_(5B)), —OC(O)R_(5B), —OC(O)OR_(5B), —OC(O)NR₂R_(5B),—NR₂C(O)R_(5B), or —NR₂C(O)OR_(5B);

R4 is H, —N(R₂)₂, optionally substituted C₁₋₃-alkyl, optionallysubstituted C₁₋₃-alkoxy, cyano, halo, hydroxy, nitro, or mono-, di-, ortrifluoromethyl;

R_(5B) is —(C₀₋₄-alkyl)-L, wherein L is a leaving group;

X is independently O, S, or N(R₂); and

R₈ is C₁₋₃ alkyl substituted by at least one —OH, —COOH, —C(O)O—C₁₋₄alkyl, C(O)N(R₂)₂, or C₃₋₅ cycloalkyl;

or a pharmaceutically acceptable salt form thereof.

Compounds of this structure, as well as processes of synthesizingcompounds of this structure are described in U.S. Patent ApplicationPublication No. US 2014-0309246, published Oct. 16, 2014, the entirecontent of which is incorporated herein by reference.

In some embodiments, the erbB inhibitor has the structure:

Compounds of these structures, as well as processes of synthesizingcompounds of these structures are described in U.S. Patent ApplicationPublication No. US 2014-0309246, published Oct. 16, 2014, the entirecontent of which is incorporated herein by reference.

Aspects of the present invention relate to a composition for thetreatment of a subject afflicted with cancer, comprising i) an erbBinhibitor; and ii) interferon-gamma (IFNγ), and a pharmaceuticallyacceptable carrier.

Aspects of the present invention also relate to a composition forsensitizing cancer to radiation or a chemotherapeutic agent, comprisingi) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention relate to a composition for preventingthe development of a tumor in a subject at risk of developing the tumor,comprising i) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention also relate to a composition forsensitizing a tumor to radiation or a chemotherapeutic agent, comprisingi) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Aspects of the present invention relate to a combination for thetreatment of a subject afflicted with cancer or preventing thedevelopment of a tumor in a subject at risk of developing the tumor,comprising i) an erbB inhibitor; and ii) interferon-gamma (IFNγ), and apharmaceutically acceptable carrier.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

Terms

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs.

As used herein, and unless stated otherwise or required otherwise bycontext, each of the following terms shall have the definition set forthbelow.

As used herein, “about” in the context of a numerical value or rangemeans ±10% of the numerical value or range recited or claimed, unlessthe context requires a more limited range.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg/kg/day” is a disclosure of 0.2mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/dayetc. up to 5.0 mg/kg/day.

As used herein, the terms “erbB-associated cancer” and “erbB-associatedtumors” are meant to refer to cancer cells and neoplasms which express amember of the erbB gene family, the expression of which results inerbB-mediated transformation.

As used herein “p185” and “p185her2/neu” refer to the erbB2 protein of185,000 molecular weight. “Neu” or “Her2” or “erbB2” or “erbB2/Her2/neu”refer to the gene that encodes the p185her2/neu protein.

P185her2/neu-associated tumors and EGFR-associated tumors are examplesof erbB-associated tumors. As used herein, the terms“erbB2/Her2/neu-associated cancer” “erbB2/Her2/neu associated tumors”and “p185her2/neu-associated cancer” are meant to refer to cancer cellsand neoplasms which express p185her2/neu. ErbB2/Her2/neu-associatedcancer is an erbB associated cancer in which the cellular transformationis mediated by tyrosine kinase activity related to p185her2/neu.

As used herein, the terms “EGFR-associated cancer” and “EGFR-associatedtumors” are meant to refer to cancer cells and neoplasms which expressEGFR. EGFR-associated cancer is an erbB-associated cancer in which thecellular transformation is mediated by tyrosine kinase activity relatedto EGFR.

p185her2/neu is also described in U.S. Pat. No. 7,625,558, issued Dec.1, 2009, and U.S. Patent Application Publication No. 2012/0164066,published Jun. 28, 2012, the entire content of each of which isincorporated herein by reference.

As used herein, an “anti-p185her2/neu polypeptide” is a polypeptide thatis capable of specifically binding to p185her2/neu. In some embodiments,the anti-p185her2/neu polypeptide specifically binds to p185her2/neu,but is incapable of binding to an erbB family protein other thanp185her2/neu under cell culture or physiological conditions. In someembodiments, an anti-p185her2/neu polypeptide is capable of binding top185her2/neu such that it significantly inhibits (either partially orcompletely) a biological activity of p185her2/neu. In some embodiments,the biological activity of p185her2/neu is dimerization with anotherp185her2/neu protein, or another erbB family protein. In someembodiments, the biological activity of p185her2/neu is tyrosine kinaseactivity. In some embodiments, an anti-p185her2/neu polypeptide iscapable of binding to p185her2/neu, without significantly inhibiting abiological activity of p185her2/neu.

As used herein, an “anti-EGFR polypeptide” is a polypeptide that iscapable of specifically binding to EGFR. In some embodiments, theanti-EGFR polypeptide specifically binds to EGFR, but is incapable ofbinding to an erbB family protein other than EGFR under cell culture orphysiological conditions. In some embodiments, an anti-EGFR polypeptideis capable of binding to EGFR such that it significantly inhibits(either partially or completely) a biological activity of EGFR. In someembodiments, the biological activity of EGFR is dimerization withanother EGFR protein, or another erbB family protein. In someembodiments, the biological activity of EGFR is tyrosine kinaseactivity. In some embodiments, an anti-EGFR polypeptide is capable ofbinding to EGFR, without significantly inhibiting a biological activityof EGFR.

In some embodiments, peptides which mimic antibodies are provided toinhibit multimeric ensemble formation and the elevated kinase activityassociated which such formation. For example, peptides are designedwhich have sequences corresponding to CDR regions from antibodies.Methods of making such peptides are also described in Ser. No.08/257,783 filed Jun. 10, 1994 and PCT Application No. PCT/US95/07157filed Jun. 6, 1995 which is incorporated herein by reference.Peptidomimetics of antibodies against p185her2/neu are described in U.S.Pat. No. 5,663,144 issued Sep. 2, 1997, which is incorporated herein byreference.

As used herein, the term “cytotoxic” agent refers to an agent thatinhibits the biological processes of a cell, or reduces the viability orproliferative potential of a cell. As used herein, the term or“cytostatic” agent refers to an agent that inhibits the proliferativepotential of a cell. In some embodiments, cytostatic agent inhibits theproliferation of a cancer cell or a cell other than a cancer cell.Cytotoxic or cytostatic agents can function in a variety of ways, forexample, but not by way of limitation, by inducing DNA damage, inducingcell cycle arrest, inhibiting DNA synthesis, inhibiting transcription,inhibiting translation or protein synthesis, inhibiting cell division,or inducing apoptosis. As used herein, the term “chemotherapeutic agent”refers to cytotoxic, cytostatic, and antineoplastic agents thatpreferentially kill, inhibit the growth of, or inhibit the metastasis ofneoplastic cells or disrupt the cell cycle of rapidly proliferatingcells. Chemotherapeutic agents include, but are not limited to,synthetic compounds, natural and recombinant bacterial toxins, naturaland recombinant fungal toxins, natural and recombinant plant toxins,fissionable nuclides, and radionuclides. Specific examples ofchemotherapeutic agents include, but are not limited to, pokeweedantiviral protein, abrin, ricin and each of their A chains, momordin,saporin, bryodin 1, bouganin, gelonin, Diphtheria toxin, Pseudomonasexotoxin, Shiga toxin, calicheamicin, maytansinoid, lead-212,bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186,rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77,indium-111, boron-10, actinide, altretamine, actinomycin D, plicamycin,puromycin, gramicidin D, doxorubicin, colchicine, cytochalasin B,cyclophosphamide, emetine, maytansine, amsacrine, platinum-basedchemotherapeutic agents including but not limited to cisplastin andcarboplatin, etoposide, etoposide orthoquinone, teniposide,daunorubicin, gemcitabine, doxorubicin, mitoxantraone, bisanthrene,Bleomycin, methotrexate, vindesine, adriamycin, vincristine,vinblastine, BCNU, taxanes including but not limited to paclitaxel,docetaxel and cabazitaxel, tarceva, avastin, mitomycin, 5-fluorouracil,cyclophosphamide and certain cytokines such as TNF-alpha and TNF-beta.

In some embodiments, radiation therapy may commence any time after asufficient amount of time has elapsed for an active agent or agents toact on cancer or other cells in a subject. Generally, the subject isexposed to radiation in some cases 1-10 minutes after, in some cases1-10 hours after, and in some cases up to 24-72 hours afteradministration of the active agent(s). In some cases, the radiation isprovided in a single dose while in some embodiments, multiple doses areadministered over several hours, days and/or weeks. The active agentrenders the radiation resistant tumor cells radiation sensitive. Thus,once the active agent inhibits the kinase activity, exposure toradiation may follow suit. Gamma radiation is delivered according tostandard radiotherapeutic protocols using standard dosages and regimens.The administration of the active agent(s) renders the radiation moreeffective in eradicating tumor cells. Active agents of the presentinvention include fusion proteins of the present invention,anti-p185her2/neu antibodies, anti-EGFR antibodies, andinterferon-gamma.

As in the case of radiation therapy, in some embodiments chemotherapymay commence any time after a sufficient amount of time has elapsed foran active agent or agents to act on cancer or other cells in a subject.Generally, the subject is administered the chemotherapeutic in somecases 1-10 minutes after, in some cases 1-10 hours after, and in somecases up to 24-72 hours after administration of the 45 kinase inhibitingactive agent(s). In some cases, the chemotherapeutic is provided in asingle dose while in some embodiments, multiple doses are administeredover several hours, days and/or weeks. The active agent(s) renders thetumor cells more sensitive to cytotoxic agents. Thus, once the activeagent inhibits, e.g., the kinase activity, administration ofchemotherapeutics may follow suit. Chemotherapeutics are deliveredaccording to standard radiotherapeutic protocols using standard agents,dosages and regimens. In some embodiments, chemotherapy and radiationtreatments are both employed following the administration of the activeagent(s). Active agents of the present invention include fusion proteinsof the present invention, anti-p185her2/neu antibodies, anti-EGFRantibodies, and interferon-gamma.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient, slowing in therate of degeneration or decline, making the final point of degenerationless debilitating, improving a subject's physical or mental well-being,or prolonging the length of survival. The treatment or amelioration ofsymptoms can be based on objective or subjective parameters; includingthe results of a physical examination, neurological examination, and/orpsychiatric evaluations.

“Effective amount” and “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a fusion protein, anantibody, antigen-binding fragment, antibody composition,interferon-gamma, or a combination thereof as described herein,effective to achieve a particular biological or therapeutic result suchas, but not limited to, biological or therapeutic results disclosed,described, or exemplified herein. A therapeutically effective amount ofthe fusion protein, the antibody or antigen-binding fragment thereof mayvary according to factors such as the disease state, age, sex, andweight of the subject, and the ability of the antibody orantigen-binding fragment thereof to elicit a desired response in thesubject. In embodiments of the invention, such results may include, butare not limited to, the treatment of cancer, as determined by any meanssuitable in the art.

The fusion proteins, anti-p185her2/neu antibodies, anti-EGFR antibodies,and interferon-gamma may be administered to a subject in apharmaceutically acceptable carrier or carriers. “Pharmaceuticallyacceptable” refers to those properties and/or substances which areacceptable to the subject from a pharmacological/toxicological point ofview and to the manufacturing pharmaceutical chemist from aphysical/chemical point of view regarding composition, formulation,stability, patient acceptance and bioavailability. “Pharmaceuticallyacceptable carrier” refers to a medium that does not interfere with theeffectiveness of the biological activity of the active ingredient(s) andis not toxic to the host to which it is administered.

Antibodies

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), monovalent antibodies, and multivalentantibodies. Additionally, the term “antibody” refers to all isotypes ofimmunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including variousmonomeric and polymeric forms of each isotype, unless otherwisespecified.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include but are not limited to Fv, Fab, Fab′,Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibodymolecules (e.g., scFv); and multispecific antibodies formed fromantibody fragments. Various techniques have been developed for theproduction of antibody fragments, including proteolytic digestion ofantibodies and recombinant production in host cells; however, othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In some embodiments, the antibody fragment ofchoice is a single chain Fv fragment (scFv). “Single-chain Fv” or “scFv”antibody fragments comprise the V H and V L domains of antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V Hand V L domains which enables the scFv to form the desired structure forantigen binding. For a review of scFv and other antibody fragments, seeJames D. Marks, Antibody Engineering, Chapter 2, Oxford University Press(1995) (Carl K. Borrebaeck, Ed.).

The term “epitope” refers to a portion of a molecule (the antigen) thatis capable of being bound by a binding agent, e.g., an antibody, at oneor more of the binding agent's antigen binding regions. Epitopes usuallyconsist of specific three-dimensional structural characteristics, aswell as specific charge characteristics.

As used herein, “monoclonal antibody” means an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants, each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495-97 (1975),or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The monoclonal antibodies may also be isolated from phagedisplay libraries using the techniques described, for example, inClackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol.Biol. 222 (3):581-97 (1991).

The term “hybridoma” or “hybridoma cell line” refers to a cell linederived by cell fusion, or somatic cell hybridization, between a normallymphocyte and an immortalized lymphocyte tumor line. In particular, Bcell hybridomas are created by fusion of normal B cells of definedantigen specificity with a myeloma cell line, to yield immortal celllines that produce monoclonal antibodies. In general, techniques forproducing human B cell hybridomas, are well known in the art (Kozbor etal., Immunol. Today 4:72 (1983); Cole et al., in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc. 77-96 (1985)).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

As used herein, “fully human antibody” is an antibody that is completelyhuman. A human antibody is one which possesses an amino acid sequencewhich corresponds to that of an antibody produced by a human or a humancell or derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. In someembodiments, a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. Fully human antibodiesmay be generated by, e.g., phage display, or in animals (such as mice)which have been genetically engineered to produce human antibodies.Exemplary methods of producing fully human antibodies are described inU.S. Pat. Nos. 7,414,170; 7,803,981; in U.S. Patent Application No.2008/0248531, and in McCafferty et al., “Phage antibodies: filamentousphage displaying antibody variable domains” Nature (1990) 348 (6301):552-554; Osbourn J K, “Proximity-guided (ProxiMol) antibody selection”Methods Mol. Biol. (2002) 178: 201-5; and Lonberg et al., “Humanantibodies from transgenic mice” Int. Rev. Immunol. (1995) 13(1):65-93,the contents of each of which are hereby incorporated by reference intheir entireties.

“Humanized antibodies” means antibodies that contain minimal sequencederived from non-human immunoglobulin sequences. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a hyper variable region of the recipient arereplaced by residues from a hypervariable region of a non-human species(donor antibody) such as mouse, rat, rabbit or nonhuman primate havingthe desired specificity, affinity, and capacity. See, for example, U.S.Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, eachherein incorporated by reference. In some instances, framework residuesof the human immunoglobulin are replaced by corresponding non-humanresidues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762, each herein incorporated by reference). Furthermore,humanized antibodies may comprise residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance (e.g., to obtain desiredaffinity). In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details see Joneset al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27(1988); and Presta, Curro Opin. Struct. Biol. 2:593-96 (1992), each ofwhich is incorporated herein by reference.

Antibodies of the invention also include antibodies produced in anon-human mammalian host, more particularly a transgenic mouse,characterized by inactivated endogenous immunoglobulin (Ig) loci. Insuch transgenic animals, competent endogenous genes for the expressionof light and heavy subunits of host immunoglobulins are renderednon-functional and substituted with the analogous human immunoglobulinloci. These transgenic animals produce human antibodies in thesubstantial absence of light or heavy host immunoglobulin subunits. See,for example, U.S. Pat. No. 5,939,598, the entire contents of which areincorporated herein by reference.

Those skilled in the art will be aware of how to produce antibodymolecules of the present invention. For example, polyclonal antisera ormonoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the protein which elicits an antibody response in the mammal.For instance, a mammal can be immunized with irradiated cells that weretransfected with a nucleic acid encoding the protein such that highlevels of the protein were expressed on the cell surface. The progressof immunization can be monitored by detection of antibody titers inplasma or serum. Standard ELISA or other immunoassay can be used withthe immunogen as antigen to assess the levels of antibodies. Followingimmunization, antisera can be obtained, and, if desired IgG moleculescorresponding to the polyclonal antibodies may be isolated from thesera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art. Hybridoma cells can be screened immunochemically for productionof antibodies which are specifically reactive with the oligopeptide, andmonoclonal antibodies isolated.

Stretches of Consecutive Amino Acid Sequences

Described herein are fusion proteins comprising stretches of consecutiveamino acid sequences that can not only bind to a particular antigen, butcan also bind to antibodies and comprise a biologically active portionof interferon-gamma. In some embodiments, the fusion protein bindsp185her2/neu. In some embodiments, the fusion protein binds EGFR. Suchfusion proteins can have at least one protein segment that is capable ofbinding to the Fc region of an antibody. In some embodiments, theFc-binding segment can be contiguous with an antigen-specific peptide.Such an antigen-binding protein can be produced by the combination of aprotein derived from a portion of Protein A, a Staphylococcus aureuscell wall component that has the ability to bind to certain antibodyisotypes, with an antigen-specific peptide. In one embodiment, this typeof antigen-binding fusion protein comprises a ZZ polypeptide (SEQ ID NO.4), derived from a portion of Protein A, linked to AHNP. The fusionprotein may also include another stretch of consecutive amino acids thathas the sequence of at least a portion of interferon-gamma. A ZZpolypeptide can be linked to an antibody fragment to function as anFc-binding domain. For example, a ZZ polypeptide could be linked to anantibody-derived fragment single chain Fv (scFv) to allow the scFv tointeract with the Fc portion of an antibody. Similarly, ZZ polypeptidecould be linked to an interferon-gamma to allow the interferon-gamma tointeract with the Fc portion of an antibody. In some embodiments, theability of the fusion protein to interact with antibodies may allow forindirect interaction with Fc receptors via the constant region of theantibody.

Indirect linkage can be mediated by an “oligopeptide linker” such aspoly-glycine or a glycine-serine oligopeptide, for example, GGGGS (SEQID NO: 6) or GGGGGS (SEQ ID NO: 7). Other such linkers are known in theart and should be considered to be encompassed by this term. (Robinsonand Sauer, 95 PNAS 5929-34 (1998), Tang et al., 271(26) J. Bio. Chem.15682-86 (1996). In addition, the various components of the fusionproteins described herein can be directly linked to one another bysplicing together their respective gene segments via genetic engineeringtechniques well known in the art. In general, an oligopeptide linker ofthe invention will range from 5 to 50, from 5 to 30, from 10-30, or from12-25 amino acids in length.

In addition to the stretches of consecutive amino acid sequencesdescribed herein, it is contemplated that variants thereof can beprepared by introducing appropriate nucleotide changes into the encodingDNA, and/or by synthesis of the desired consecutive amino acidsequences. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the stretches ofconsecutive amino acids described herein when expression is the chosenmethod of synthesis (rather than chemical synthesis for example), suchas changing the number or position of glycosylation sites or alteringthe membrane anchoring characteristics.

Variations in the sequences described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the consecutive amino acid sequence ofinterest that results in a change in the amino acid sequence as comparedwith the native sequence. Optionally the variation is by substitution ofat least one amino acid with any other amino acid in one or more of thedomains. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence with that of homologousknown protein molecules and minimizing the number of amino acid sequencechanges made in regions of high homology. Amino acid substitutions canbe the result of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, such as the replacementof a leucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence. It is understood that anyterminal variations are made within the context of the inventiondisclosed herein.

Amino acid sequence variants of the a protein, such as a fusion protein,are prepared with various objectives in mind, including increasing theaffinity of the fusion for p185her2/neu or EGFR, facilitating thestability, purification and preparation of the fusion protein, modifyingits plasma half life, improving therapeutic efficacy, and lessening theseverity or occurrence of side effects during therapeutic use of thefusion protein.

Amino acid sequence variants of these sequences are also contemplatedherein including insertional, substitutional, or deletional variants.Such variants ordinarily can prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the target-binding monomer, by which DNAencoding the variant is obtained, and thereafter expressing the DNA inrecombinant cell culture. Fragments having up to about 100-150 aminoacid residues can also be prepared conveniently by in vitro synthesis.Such amino acid sequence variants are predetermined variants and are notfound in nature. The variants exhibit the qualitative biologicalactivity (including target-binding) of the nonvariant form, though notnecessarily of the same quantative value. While the site for introducingan amino acid sequence variation is predetermined, the mutation per seneed not be predetermined. For example, in order to optimize theperformance of a mutation at a given site, random or saturationmutagenesis (where all 20 possible residues are inserted) is conductedat the target codon and the expressed variant is screened for theoptimal combination of desired activities. Such screening is within theordinary skill in the art.

Amino acid insertions usually will be on the order of about from 1 to 10amino acid residues; substitutions are typically introduced for singleresidues; and deletions will range about from 1 to 30 residues.Deletions or insertions preferably are made in adjacent pairs, i.e. adeletion of 2 residues or insertion of 2 residues. It will be amplyapparent from the following discussion that substitutions, deletions,insertions or any combination thereof are introduced or combined toarrive at a final construct.

In an aspect, the invention concerns a compound comprising a stretch ofconsecutive amino acids having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to an amino acidsequence disclosed in the specification, a figure, or a SEQ ID NO. ofthe present application.

The % amino acid sequence identity values can be readily obtained using,for example, the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)).

Fragments of native sequences are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Again, it is understood that any terminal variations are made within thecontext of the invention disclosed herein. Certain fragments lack aminoacid residues that are not essential for a desired biological activityof the sequence of interest.

Any of a number of conventional techniques may be used. Desired peptidefragments or fragments of stretches of consecutive amino acids may bechemically synthesized. An alternative approach involves generatingfragments by enzymatic digestion, e.g. by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredpolypeptide/sequence fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Ala (A) val; leu; ile val Arg (R)lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C)ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H)asn; gin; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leuLeu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of thesequence are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro;

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)) or other known techniques can be performedon the cloned DNA to produce the variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,Science, 244:1081-1085 (1989)). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Polynucleotides and Expression

The fusion proteins described herein can be made by recombinantprocesses and, therefore, may include amino acid sequences derived frommore than one species (i.e. chimeric constructs) or may be engineered tohave a human, or human-like, amino acid composition (i.e., a humanizedconstruct). Accordingly, provided herein are vectors comprisingpolynucleotides capable of encoding the described fusion proteins. Thevectors can be expression vectors. Recombinant expression vectorscontaining a sequence encoding a polypeptide of interest are thusprovided. The expression vector may contain one or more additionalsequences such as, but not limited to, regulatory sequences (e.g.,promoter, enhancer), a selection marker, and a polyadenylation signal.Vectors for transforming a wide variety of host cells are well known tothose of skill in the art. They include, but are not limited to,plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs), aswell as other bacterial, yeast and viral vectors. The vectors describedherein may be integrated into the host genome or maintainedindependently in the cell or nucleus.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences can be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression/production of an antibody orantigen-binding fragment can be within the cytoplasm of the cell, and/orinto the extracellular milieu such as the growth medium of a cellculture.

Recombinant expression vectors contemplated to be within the scope ofthe description include synthetic, genomic, or cDNA-derived nucleic acidfragments that encode at least one recombinant protein which may beoperably linked to suitable regulatory elements. Such regulatoryelements may include a transcriptional promoter, sequences encodingsuitable mRNA ribosomal binding sites, and sequences that control thetermination of transcription and translation. Expression vectors,especially mammalian expression vectors, may also include one or morenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, other 5′ or 3′flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences(such as necessary ribosome binding sites), a polyadenylation site,splice donor and acceptor sites, or transcriptional terminationsequences. An origin of replication that confers the ability toreplicate in a host may also be incorporated. Such vectors may beintegrated into the host genome or maintained independently in the cellor nucleus.

The vectors described herein can be used to transform various cells withthe genes encoding the disclosed fusion proteins. For example, thevectors may be used to generate scaffold or antigen-bindingprotein-producing cells or cell lines. Thus, another aspect featureshost cells transformed with vectors comprising a nucleic acid sequenceencoding a fusion protein. The host cells disclosed herein can beprokaryotic or eukaryotic cells, For example the host cell can be abacteria. In a preferred embodiment, the bacterial host cell is E. coli.Of course, the host cell can also be a mammalian cell, such as a Chinesehamster ovary (CHO) cell line. Numerous other such host cells,prokaryotic and eukaryotic, are known in the art and are considered tobe within the scope of this disclosure.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the inventive methods, in accordance with thevarious embodiments described and exemplified herein. The technique usedshould provide for the stable transfer of the heterologous gene sequenceto the host cell, such that the heterologous gene sequence is heritableand expressible by the cell progeny, and so that the necessarydevelopment and physiological functions of the recipient cells are notdisrupted. Techniques which may be used include but are not limited tochromosome transfer (e.g., cell fusion, chromosome mediated genetransfer, micro cell mediated gene transfer), physical methods (e.g.,transfection, spheroplast fusion, microinjection, electroporation,liposome carrier), viral vector transfer (e.g., recombinant DNA viruses,recombinant RNA viruses) and the like. Calcium phosphate precipitationand polyethylene glycol (PEG)-induced fusion of bacterial protoplastswith mammalian cells can also be used to transform cells.

It is fully contemplated that the vectors such as those described hereincan be used to transform prokaryotic and/or eukaryotic cells tofacilitate expression of the described fusion proteins. In someembodiments the described vectors are used to facilitate fusion proteinexpression in bacteria, such as E. coli. While any E. coli strain can beused to express the proteins described herein, some preferred strainsinclude: BL21 (DE3), BL21-CodonPlus® (DE3)-RP, BL21-Codon Plus®(DE3)-RIL, BL21-(DE3)-pLysS (Stratagene). Eukaryotic cells can also beused with vectors to facilitate protein expression. While those of skillin the art will recognize that a wide variety of eukaryotic cells willbe suitable for this purpose, some preferred embodiments includemammalian cells and insect cells. For example, in one embodiment Chinesehamster ovary (CHO) cells can be used with the vectors to facilitateexpression of the fusion protein constructs provided herein. Inalternative embodiments, insect cells, such as Sf9 cells or S2 cells,can be used to with the described vectors to facilitate expression ofthe protein constructs provided herein. Furthermore, those of skill inthe art will understand that vectors, not expressly disclosed herein,can be used for the same purpose of expressing, or replicating nucleicacids encoding, the described antigen binding proteins.

The described fusion proteins can be encoded by a variety ofpolynucleotides capable of encoding the amino acid sequences providedherein. These polynucleotides can also be incorporated into vectorsuseful for the maintenance, replication, and/or expression of thepolynucleotides encoding the described antigen-binding proteins or thedescribed portions thereof. The vectors described above can be used toengineer cells to express the antigen-binding proteins or the describedportions thereof encoded by the polynucleotides disclosed herein.

Compositions

Also described herein are compositions containing a fusion protein orfusion proteins of the invention and a pharmaceutically acceptablecarrier. Such compositions can be used to administer the describedfusion proteins to a subject or store or to maintain the describedfusion proteins. Any of the described fusion proteins can be used toproduce such compositions, which may include more than one of thedisclosed proteins. In addition, such compositions can include otheragents, such as therapeutic agents, preservatives, antimicrobial agents,and the like.

Described herein are compositions comprising at least one disclosedprotein and a pharmaceutically acceptable carrier. The compositions canbe formulated as any of various preparations that are known and suitablein the art, including those described and exemplified herein. In someembodiments, the compositions are aqueous formulations. Aqueoussolutions can be prepared by admixing the antigen-binding proteins inwater or suitable physiologic buffer, and optionally adding suitablecolorants, flavors, preservatives, stabilizing and thickening agents andthe like as desired. Aqueous suspensions can also be made by dispersingthe antigen-binding proteins in Water or physiologic buffer with viscousmaterial, such as natural or synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are liquid formulations and solid form preparations whichare intended to be converted, shortly before use, to liquidpreparations. Such liquids include solutions, suspensions, syrups,slurries, and emulsions. Liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogennated edible fats or oils); emulsifying agents (e. g., lecithinor acacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). These preparations maycontain, in addition to the active agent, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The compositions may bein powder or lyophilized form for constitution With a suitable vehiclesuch as sterile water, physiological buffer, saline solution, oralcohol, before use.

The compositions can be formulated for injection into a subject. Forinjection, the compositions described can be formulated in aqueoussolutions such as water or alcohol, or in physiologically compatiblebuffers such as Hanks's solution, Ringer's solution, or physiologicalsaline buffer. The solution may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Injection formulationsmay also be prepared as solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations suitable forinjection, for example, by constitution with a suitable vehicle, such assterile water, saline solution, or alcohol, before use.

The compositions can be formulated in sustained release vehicles ordepot preparations. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

The proteins described herein may be administered orally in anyacceptable dosage form such as capsules, tablets, aqueous suspensions,solutions or the like. The proteins may also be administeredparenterally including but not limited to: subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intranasal, topically, intrathecal, intrahepatic, intralesional, andintracranial injection or infusion techniques. Generally, the proteinswill be intravenously or intraperitoneally, for example, by injection.

The subject can be any animal, and preferably is a mammal such as amouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, donkey, cow,horse, pig, and the like. In some embodiments, the mammal is a human.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

The present invention is not intended to be limited by any theory. Thisinvention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrativeof the invention as defined in the claims which follow thereafter.

EXPERIMENTAL DETAILS

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1

Enhanced Activity of 7.16.4 and IFN-γ on Tumors Transformed by OncogenicNeu.

p185her2/neu is a member of the ERBB family of receptor tyrosine kinasesand has been validated as a clinical target for breast and stomachcancers. Monoclonal antibodies to the oncoprotein of the rat origin weredeveloped to establish the foundation for targeted therapies to solidtumors (Drebin et al., 1986; Drebin et al., 1984). One of the monoclonalantibodies, mAb7.16.4, has a shared epitope with trastuzumab, a FDAapproved therapeutic agent in clinical use (Zhang et al., 1999). 7.16.4is active on Erbb2/neu transformed rodent and human tumors in a varietyof assays (Cai et al., 2013; Zhang et al., 1999). 7.16.4 has been usedin many labs around the world in transgenic animal models of tumorsinduced by the neu oncogene (Katsumata et al., 1995; Park et al., 2010;Stagg et al., 2011).

Recent studies defined a role for CD8 IFN-γ secreting cells and NK ADCCmediating cells as contributory elements in 7.16.4 therapy of implantedtumor model of neu transformed cells (Park et al., 2010; Stagg et al.,2011). It is expected that both active innate and adaptive immune cellsthat translocate and reside in the tumors contribute to the anti-canceractivity of 7.16.4 and trastuzumab.

Experiments were conducted to examine whether IFN-γ could directlyenhance 7.16.4 anti-tumor activity. The target tumor cell line H2N113was established from the tumor of MMTV-neu transgenic mice (Stagg etal., 2011). In vitro proliferation assay demonstrated that 7.16.4 couldinhibit H2N113 in a dose-dependent manner (Du et al 2013). H2N113 tumorcells were implanted into BALB/c-MMTV-neu mice as reported previously(Stagg et al., 2011). Treatment began when tumors were apparent (10 daysafter tumor inoculation). Mice were treated with a control antibody,IFN-γ alone, sub-optimal amount of 7.16.4 (5 mg/kg), or the combinationof 7.16.4 and IFN-γ. As shown in FIG. 1 , the combination group(7.16.4+IFN) showed dramatically suppressed tumor growth. The dataherein indicate that IFN-γ enhances the effectiveness ofanti-p185her2/neu antibody targeted therapy.

Example 2

Activity of 4D5scFv-IFNγ Fusion Protein on Tumors Transformed by ErbB2.

Previously a novel “Grababody” approach to empower a scFv construct withimmune cell functions was reported (Cai, 2013) (disclosed in UPN-5599).This approach utilizes the IgG binding Z domain placed on the C-terminusto the scFv to capture endogenous circulating IgG. Using the scFv top185her2/neu as an example, it was shown that the Grababody 4D5scFvZZ(SEQ ID NO: 1) binds to the target receptor on the tumor cells anddemonstrates CDC and ADCC activity towards antigen-positive tumor cells.Most importantly, in the in vivo xenograft mice model, the Grababodysignificantly reduces the growth of the malignant tumors transformed byErbB2/Her2/neu (Cai, 2013).

To show that IFN-γ can improve the activity of anti-tumor activity ofscFv based Grababody 4D5scFvZZ (SEQ ID NO: 1), a single chain fusionmolecule 4D5scFvZZ-IFNγ was constructed (SEQ ID NO: 2). The anti-tumoractivity of 4D5scFvZZ (SEQ ID NO: 1) and 4D5scFvZZ-IFNγ (SEQ ID NO: 2)was compared using the T6-17 in vivo model in nude mice. Athymic nudemice were inoculated with 5×10⁵ T6-17 cells. Mice carrying tumorreceived 4D5scFvZZ (SEQ ID NO: 1), 4D5scFvZZ-IFNγ (SEQ ID NO: 2) orcontrol buffer at the dose of 7 mg/kg, three times per week via i.p.injection. As shown in FIG. 2A-FIG. 2B, 4D5scFvZZ-IFNγ (SEQ ID NO: 2)has better activity than 4D5scFvZZ (SEQ ID NO: 1) to limit the growth ofT6-17 tumors.

Binding of 4D5scFv-ZZ-IFNγ and 4D5scFv-IFNγ to p185her2/Neu ExpressingCells.

It was previously shown that the 4D5scFv in the Grababody 4D5scFv-ZZ wasactive to bind p185her2/neu that was either immobilized on the chip orexpressed on the cell surface (Cai, 2013). To confirm that the4D5scFv-ZZ-IFNγ and 4D5scFv-IFNγ are also corrected folded and containactive 4D5scFv unit, FACS binding assays on p185her2/neu-expressingT6-17 cells were performed. As shown in FIG. 3 , both constructs wereable to bind T6-17.

Verification of the Fusion Proteins for IFNγ Activity

IFNγ is known to be able to induce class I MHC antigen expression intumor cells. Activity on MHC expression in SKBR3 cells was examined toverify that the IFNγ unit in the fusion protein is active. As shown inFIG. 4 , the recombinant protein 4D5scFv-ZZ-IFNγ demonstrated class IMHC-stimulating activity comparable to a free IFNγ molecule. Both IFNγand 4D5scFv-ZZ-IFNγ had no effect on class II MHC antigen.

Comparison of In Vivo Activity of 4D5scFv-ZZ-IFNγ and 4D5scFv-IFNγ.

Experiments using T6-17 in vivo model demonstrated that 4D5scFv-ZZ-IFNγhad better activity than 4D5scFv-IFNγ (FIG. 5 ).

IFNα Appears not to have the Same Activity as IFNγ to FacilitateAnti-p185her2/Neu Antibody.

IFNα appears to have anti-tumor activity on its own in the in vivo tumormodel but it could not enhance mAb 7.16.4 activity to suppress thegrowth of xenografted tumors (FIG. 7 ).

Effect of Co-Treatment on MDSC

Myeloid-derived suppressor cells (MDSC) are defined as CD45+ cells thatare also Cd11b+ and GR-1+. MDSC were isolated from tumors at the end oftreatments. As shown in FIG. 8 , the co-treatment with 7.16.4 and IFNγled to the most reduction of MDSC populations in tumor tissues ofxenografted mice. This study indicates that the co-treatment may preventthe infiltration of certain immune suppressor cells into the tumormicroenvironment (FIG. 8 ).

An in vitro cell migration experiment was also performed. H2N113 cellswere treated with control, 7.16.4, IFNγ, or 7.16.4 plus IFNγ for 3 days.The same treatments were also incubated in blank wells without anyH2N113 cells as controls. The conditioned medium from each wells werecollected and placed into the bottom chamber of a cell migration device.MDSC isolated from mice were placed into the upper chamber. Cellsmigrated from the upper chamber to the lower one were counted. As shownin FIG. 9 , H2N113 conditioned medium clearly attracted MDSC cells tomigrate into the lower chamber. 7.16.4 slightly reduced the migration,but the co-treatment of 7.16.4 and IFNγ completely blocked MDSCmigration induced by H2N113. This study suggests that the co-treatmentaffect the tumor cells and prevent tumor cells from attracting immunesuppressor cells.

Example 3

Disabling the erbB2 Kinase Reverses Features of the Malignant Phenotypeto Permit Interferon-γ to Act on Human Breast Cancer Cells.

This study provides additional biochemical studies defining the effectsof ordered therapy of disabling erbB kinases with either monoclonalantibody or small molecules followed or concomitant with interferongamma. The data is remarkably clear the there are two steps tophenotypic reversion—one is diminution of malignant properties thataccompany disabling of either erbB2/neu or erbB1 kinases. Thecombination action of antierbB2 and interferon gamma leads todegradation of Snail, which is dependent on GSK3 (and can be abolishedby a pharmacological inhibitor. Secondly the phenotypically revertedcell can only then be acted on by interferon gamma (and not any otherinterferon). This is new and profound observation that explains optimaluse of these reagents to alter malignant properties and tumor survival.

SUMMARY

erbB2 confers transforming properties and is amplified in approximately30% of breast cancers. One therapeutic approach to dampen erbB2signaling is monoclonal antibody (mAb) targeting. Although direct actionof targeting antibodies results in erbB2 down-modulation and phenotypereversal, use in vivo suggested a contribution of immune cells andcytokines. Unfortunately, mAb-based therapy often is not curative andtumors reoccur, which may be the consequence of cells that self-renewand resist therapy. The data herein show anti-erbB2 mAb concurrent withIFN-γ reverses the malignant phenotype beyond either treatment alone.Exposure of breast cancer cells to anti-erbB2 mAb and IFN-γ reduces thetranscriptional repressor snail through accentuated GSK3-β activity.These data identify a mechanism for co-treatment and support an approachfor developing therapeutics.

Reversion of the malignant phenotype of erbB2-transformed cells can bedriven by anti-erbB2/neu monoclonal antibodies (mAb) which disrupt thereceptor's kinase activity. The biologic effects of IFN-γ withanti-erbB2/neu mAb was examined on erbB2-positive cells. IFN-γ had noeffect on its own. In contrast, treatment of the tumors withanti-erbB2/neu mAb followed by IFN-γ led to dramatic inhibition of tumorgrowth in vitro and in vivo with minimal mAb dosing and enhanced theeffects of chemotherapy. IFN-γ with mAb treatment of IFNγR knock downtumors did not show combination eradication effects, indicating INF-γacts dominantly on the tumor itself. mAb and IFN-γ decreased Snailexpression in tumor cells, reflecting loss of stem cell-like propertiesthrough enhanced activity of GSK3-β and KLF4.

Significance

Monoclonal antibody-based targeted therapy of erbB driven tumorsbenefits patient outcome in multiple cancers; however, some patients donot respond, and virtually all responders eventually relapse. Theexperiments described herein found that interferon-gamma (IFN-γ) isessential to modifying intrinsic properties of transformed cells thathave undergone phenotypic reversion with p185 kinase disablinganti-erbB2 mAb. These experiments establish that IFN-γ concurrent withor following anti-erbB2 mAb inhibits a vital intrinsic tumor-signalingpathway limiting stem cell-like properties and has a combination effectthat provides optimal therapeutic effects on erbB2-transformed humanbreast cancer cells. Without wishing to be bound by any scientifictheory, the data herein suggest that co-administration or sequentialordering of anti-erbB2 mAb with IFN-γ establishes a set of changes ofintrinsic cellular phenotype. The present invention provides thiscombination treatment for therapies as disclosed herein.

Co-administration or sequential ordering of anti-erbB2 mAb with IFN-γmay greatly reduce the need for the mAb components and genotoxicchemotherapeutics necessary for treatment of humans with erbB2-drivencancers.

Highlights

-   -   IFN-γ and 4D5 act directly on HER2-positive breast cancer cells    -   Disabling HER2 signaling preferentially inactivates the        PI-3K/Akt pathway    -   IFN-γ, but not IFN-β, cooperates with 4D5 directly on HER2+        breast cancer cells    -   IFN-γ and 4D5 alters KLF4 levels and degrades Snail by        GSK3-β/proteasome pathway.    -   Treatment with IFN-γ and 4D5 degrades snail through the        GSK3-β/proteasome pathway    -   Combination treatment could reduce the amount of targeted mAb        needed in vivo.

Introduction

The erbB or HER family of receptor tyrosine kinases consists of erbB1(the epidermal growth factor receptor (EGFR)/HER1), erbB2(p185/neu/HER2), erbB3 (HER3), and erbB4 (HER4), which can formhomomeric and heteromeric assemblies (Kokai et al., 1989; Qian et al.,1994). ErbB receptor tyrosine kinases participate in a variety of signaltransduction cascades, including the Ras/Raf/MEK/ERK and PI-3K/Aktpathways. ErbB2 is amplified in ˜30% of breast cancer patients, andamplification is associated with poor prognosis and decreased survival(Riemsma et al., 2012). In various cancers, amplified or mutated formsof these kinases drive increased proliferation, migration, survival,evasion of apoptosis, metastasis, and resistance to chemotherapeuticsand ionizing radiation.

Recognition that mAbs could disable the p185erbB2/HER2/neu tyrosinekinase receptor complex and also lead to reversal of the malignantphenotype challenged the dogma that transformed cells could onlyprogressively become more abnormal (Drebin et al., 1985; Schechter etal., 1984). Reversal of the malignant phenotype by anti-erbB2 mAb occurswithin 24 hours of mAb binding (Drebin et al., 1986; Lee et al., 2012;O'Rourke et al., 1997; Qian et al., 1994a) and begins with downregulation of p185erbB2/neu receptor tyrosine kinase proteins causingdiminished enzymatic activity (Drebin et al., 1988; Drebin et al., 1986;Furuuchi et al., 2007; Sliwkowski and Mellman, 2013; Wada et al., 1990;Zhang et al., 2007). All of these clinically-relevant features areimproved by anti-ErbB2 mAb therapy (Baselga et al., 2001; Hudis, 2007;Kiessling et al., 2002; Meric-Bernstam and Hung, 2006; Romond et al.,2005; Seidman et al., 2001) and more dramatically with the inclusion ofa second antibody, which more completely disables erbB2/neu.

The laboratory of Dr. Mark Greene demonstrated that disabling the kinasecomplex with monoclonal antibodies (mAb) specific for the ectodomaincould reverse aspects of the malignant phenotype in vitro and in vivo(Drebin et al., 1988a; Drebin et al., 1985; Drebin et al., 1986). Theseapproaches were advanced to the clinic and single and dual antibodytherapies are now applied to human diseases such as erbB2-positivebreast cancer (Baselga et al., 2010; Cortes et al., 2012; Portera etal., 2008). The mechanism through which anti-erbB2 mAb acts on tumors invivo is complicated, but may include elements of both the adaptive andinnate immune system for optimal activity (Park et al., 2010).Furthermore, using an implant model, Stagg and colleagues demonstrated arequirement for Type I and II interferons (IFNs) in mediating anti-erbB2mAb functions in vivo, through endowing CD8 T cell cytotoxic activities(Stagg et al., 2011). The earliest studies by the Hynes laboratoryindicated that IFN-γ could limit p185erbB2/neu expression at the mRNAlevel (Marth et al., 1990) in some tumors. IFN-γ was found to increaseerbB1 (EGFR) levels (Hamburger and Pinnamaneni, 1991) and TGF-αsecretion through increased EGFR activity (Uribe et al., 2002) as wellas to promote tumor evasion of the immune system in models of coloncarcinoma (Beatty and Paterson, 2000). IFN-γ was also one of the firstrecombinant cytokines tested as a single agent in trials of multiplehuman cancers, but it led to few if any beneficial outcomes. Thus,clinical efforts using IFN-γ alone as a therapeutic for mostmalignancies have not been pursued (Krigel et al., 1985).

Malignant evolution of tumors that resist targeted therapy involves theemergence of complex transcriptional functions resembling those of stemcells. Co-expression of erbB2 and constitutively activephosphatidylinositol-3 kinase (PI-3K-CA) produces tumors with enrichedtranscripts similar to those of stem-like cells or cells that havepassaged through an epithelial to mesenchymal transition. In addition,mammary tumors in erbB2+/PI-3K-CA transgenic mice are prone to lungmetastases. Transformed cells isolated from such tumors more readilyform mammospheres in cell culture (Hanker et al., 2013). Analysis ofmouse fetal mammary stem cells (fMaSC) revealed that these cells retainstem properties in vitro such as sphere formation and serial propagationas well as express multiple lineage markers. Comparison of fMaSC genesignatures to those of breast tumor arrays identified overlap withbasal-like and erbB2+ tumors (Spike et al., 2012). Finally, a recentarchival analysis of breast and lung cancers with p53 mutations revealedstem-like transcriptional signatures (Mizuno et al., 2010), implyingthat loss of p53 functionality allows for some features of phenotypicconversion.

The advanced tumor includes transformed, differentiated, and highlyproliferative cells that constitute the majority of the tumor, and asmall fraction composed of transformed, stem-like cells with slowerproliferation rates that are refractory to therapy and capable ofself-renewal (Wicha et al., 2006). Some of these cell types may undergochange; and Chaffer noted that mammary epithelial cells are able tospontaneously convert from differentiated to dedifferentiated stem-likecells, a property that could be further enhanced by oncogenictransformation (Chaffer et al., 2011). Oncogenes such as erbB2 and Rasdrive normal cells, including mammary epithelial cells, neurons, andastrocytes, toward a stem cell phenotype in vitro and in vivo leading tophenotypes which resemble the human pathologies that they model(Cicalese et al., 2009; Friedmann-Morvinski et al., 2012; Korkaya etal., 2008).

Certain proteins relevant to these phenotypic changes have beenidentified. The transcriptional repressor snail is essential forgastrulation and mesoderm formation during mammalian development (Carveret al., 2001). Snail levels increase in erbB2/neu-driven mammary tumorsand this promotes tumor recurrence in vivo. Further, elevated levels ofsnail are a predictor of decreased relapse-free survival in breastcancer patients (Moody et al., 2005). Slug and SOX9 transcriptionalproteins may similarly function together to induce a stem-like phenotypein mammary cells in addition to maintaining tumor and metastaticproperties (Guo et al., 2012). However, snail, but not slug, isspontaneously increased during recurrent tumor formation (Moody et al.,2005). Glycogen synthase kinase 3-beta (GSK3-β), while inactivated byAkt1, regulates snail through site-specific phosphorylation. Theseregulatory post-translational modifications alter snail's subcellularlocalization and stability. Specifically, GSK3-β phosphorylates snail onsix serine residues (serines 97, 101, 108, 112, 116, and 120)encompassing two motifs that promote translocation from the nucleus tothe cytoplasm and β-TRCP-mediated ubiquitination and degradation (Zhouet al., 2004). There has not been any description of erbB targetedimmune processes that govern Snail protein functions.

Products of activated immune cells such as IFN-γ have been shown toenhance the expression of the KLF4 transcription factor which itself canrepress Snail transcription (Feinberg et al., 2005; Yori et al., 2011).KLFs are members of the zinc finger family of transcription factors andtypically regulate critical aspects of cellular development anddifferentiation as well as aspects of cellular phenotype. KLF4 can beinduced in response to IFN-γ and can be decreased by TGF-β1 exposure.KLF4 over expression induces macrophage activation markers while KLF4knockdown markedly modulates the ability of IFN-γ to render thoseeffects. Yori et al (Yori et al., 2011) showed that transfection of KLF4attenuated primary tumor growth as well as affecting development ofmetastatic lesions due to decreased proliferation and increasedapoptosis of the transfected transformed cells.

This study describes a previously undefined mechanism by which limitingerbB2 kinase activity promotes degradation of snail protein, a processdependent on inhibiting PI3-K/Akt signaling andGSK3-R/proteasome-dependent elimination activities. Unexpectedly IFN-γwas found to contribute to phenotypic reversion and cell viability byactivating GSK3-β. However this mechanism is operative only if themalignant cells had been phenotypically modified by the actions of theanti-erbB2 mAb. The effect of mAb disabling of erbB2 is to accomplish afirst step in malignant phenotypic reversion, which sensitizes thereverted cells to be further modified by IFN-γ, but not IFN-β. Withoutwishing to be bound by any scientific theory, these findings provide anexplanation as to why anti-erbB2 mAb-mediated tumor elimination requiresIFN-γ and how this may lead to better human therapeutics.

The results described herein provide new insight into these processesand indicate that combinations of erbB2-targeted mAb and IFN-γ, but notIFN-α or β, modify Snail expression and contribute to phenotypicreversion and cell viability by altering GSK3-β activity and enhancingKLF4 expression in breast tumors.

These efforts have also been extended in vivo in therapeutic andprevention models.

Results

Sequential and Concurrent Anti-erbB2 mAb and Interferon-γ Act Directlyon erbB2+ Breast Cancer Cells

The laboratory of Dr. Mark Greene found that treatment of erbB2/neutransformed cells with anti-erbB2 mAb leads to rapid down modulation ofthe p185/erbB2/neu protein from the cell surface beginning within a fewhours of their interaction (Drebin et al., 1985; Drebin et al., 1986;Drebin et al., 1984). Disabling the p185/erbB2/neu kinase complex isaccompanied by formation of hypophosphorylated, tetrameric species thatare associated with growth inhibition and phenotypic reversion (Furuuchiet al., 2007). Further, studies from the laboratory of Dr. Mark Greeneand others (Lee et al., 2012; O'Rourke et al., 1998) indicate thatdisabling erbB receptors prior to addition of a secondary treatmentsensitizes the cells to genotoxic signals, a process that occurs within24 hours of diminishing kinase functions.

In order to establish optimal treatment conditions, treatment dosages aswell as ordered pairings with IFN-γ were tested over an eight-day timeframe. SK-BR-3 breast cancer cells, which are erbB2-positive,transformed human cells, were treated with three doses of IFN-γ, asingle dose of anti-erbB2 mAb (4D5), or control IgG (cIgG) to comparewith cells that were first exposed to IFN-γ for four days followed byIFN-γ and 4D5 for an additional four days or treated with 4D5 for fourdays followed by treatment with 4D5 and IFN-γ for an additional fourdays. Cells simultaneously exposed to both 4D5 and IFN-γ for eight dayswere included as well. Pre-treatment with 4D5 for 4 days followed by theaddition of IFN-γ at 5, 10, or 20 ng/mL produced a greater reduction incell viability (viabilities of 50.8±2.3, 47.3±3.7, and 40.9±4.3%,respectively) than pre-treatment with IFN-γ at 5, 10, or 20 ng/mLfollowed by addition of 4D5 (viabilities of 71.8±5.1, 67.7±5.5, and55.3±6.2% respectively). Prolonged co-treatment with 4D5 and IFN-γ at 5,10, or 20 ng/mL resulted in still greater reduction in cell viabilities(40.4±3.7, 39.4±3.2, and 29.9±3.8%, respectively) (FIG. 19A). Thesestudies established that first disabling the erbB2 kinase with mAbfollowed by IFN-γ or prolonged co-treatment with mAb and IFN-γ treatmentproduced the most significant viability effect on breast tumor cells.

Next, the detailed kinetics of the sensitizing effect using anintermediate dose of IFN-γ (10 ng/mL) was examined. Dramatic reductionin cell viability was noted over an eight-day time course in thepresence of 4D5, which could be augmented with the inclusion of IFN-γ(FIG. 19B). These studies, as well as the remaining cell-based studiesdescribed herein, were performed with a single treatment of IFN-γ, 4D5,or IFN-γ+4D5 administered the day following cell seeding (i.e. Day 0 onthe graph). Immortalized, but untransformed MCF10A cells were notaffected either by 4D5 alone or in combination with IFN-γ (FIG. 25 ).These latter data indicate that the combined ordered effects aremanifested only on cells that express the target oncogene and haveacquired malignant properties.

Changes in the malignant phenotype are most conclusively defined invitro by anchorage independent foci formation in soft agar assays(Montesano et al., 1977). We analyzed the formation of foci based onsize using ImageJ software (NIH). In the settings used, foci wereapparent when they approached 20 pixels and foci larger than 20 pixelsrepresent cells that exhibit transformed properties. Treatment with 4D5produced a dramatic reduction in the formation of foci in soft agarcompared to cIgG and IFN-γ alone as demonstrated by a greater percentageof foci less than 20 pixels (72±5% vs. 54±2% and 48±8%, respectively) aswell as fewer foci greater than 20 pixels (27±6% vs. 45±2% and 52±8%).Inclusion of IFN-γ with 4D5 produced a greater percentage of foci under20 pixels than 4D5 alone (81±2% vs. 72±5%) and a lesser percentage offoci greater than 20 pixels compared to 4D5 alone (18±2% vs. 27±5%)(FIG. 19C and FIG. 19D). Anchorage independent malignant features arethus most influenced by treatment with anti-erbB2 mAb with the additionof IFN-γ which then act on cells with certain reversed malignantfeatures.

Studies were initiated to examine if IFN-γ could complement anti-erbB2mAb in tumor growth experiments in rodents. To accomplish this, H2N113tumor cell lines were used (Stagg et al., 2008), which were derived fromMMTV-neu (BALB/c) transgenic mice. The 7.16.4 anti-erbB2 mAb recognizesthe rat and human p185/neu and with optimal doses (5 mg/kg)significantly inhibits p185neu driven tumor growth in rodents (Drebin etal., 1988b; Drebin et al., 1985). Importantly, in the experimentsreported here, lower doses (1.5 mg/kg) of anti-erbB2 mAb were employedto demonstrate surprisingly increased interactions. These experimentsillustrate a demonstrable effect of IFN-γ as well as 7.16.4 on breasttumor growth. However, combinatorial therapy significantly reduces tumorvolumes when compared to either alone (FIG. 26 ). Without wishing to bebound by any scientific theory, it is concluded that the phenotypicreversion activity of anti-erbB2 mAb can be improved by employing IFN-γas a modality to further alter malignant properties of transformedcells.

Phenotypic Effects of mAb and Interferon-γ on Other Cell Types

It was speculated that some erbB heteromeric tumors might be similarlysensitized for IFN-γ effects while other erbB1 homomeric tumors mightnot because of the action of IFN-γ on EGF/TGF expression (Uribe et al2002). First homomeric EGFR driven cell lines were utilized to determinewhether IFN-γ doses were compatible when disabling EGFR. EGFR drivencell lines were utilized to determine whether these are general effectson other cell types. Experiments (FIG. 19A) implied that combinedtreatments were the most efficacious; therefore, experiments wereperformed to determine if IFN-γ doses were compatible when disablingEGFR. Dose-response assays were performed using two cell linesexpressing EGFR—the epidermoid carcinoma cell line A431 and theglioblastoma cell line U87. The U87 cell line displays a much lowerexpression level of EGFR than A431 and it is unclear if EGFR solelydetermines the transformed phenotype. The U87 cell line is driven byEGFR VIII, which lacks portions of the extracellular domain of EGFR.A431 cells, which are transformed by the activity of high levels ofEGFR, responded to dose-dependent increases of IFN-γ in the presence ofthe anti-EGFR mAb C225 (FIG. 27A and FIG. 43D). U87 failed to respond toany dose of IFN-γ and C225. (FIG. 27B and FIG. 49 ).

Other tumor lines were analyzed, including tumor cell lines which lackor have normal levels of EGFR or erbB2/neu and little effects of thetargeting mAb was found with or without IFN-γ (not shown). Withoutwishing to be bound by any scientific theory, it is concluded that celllines which are transformed by erbB kinases are generally amenable tocombination therapies, while cell lines transformed by undefined geneticchanges without a dominant contribution of erbB kinases are not.

The erbB2⁺ breast cancer cell lines MDA-MB-453 and BT-474 displaydistinct phenotypes from the SK-BR-3 cells. MDA-MB-453 and BT-474viabilities were inhibited by 4D5 mAb and MDA-MB-453 cell viability wasfurther impeded by the inclusion of IFN-γ (FIG. 43 ). BT-474 cells didnot respond to this ordered approach (FIG. 43 ). A possible explanationmight be that BT-474 cells express estrogen receptors (ER). Treatmentwith the ER antagonist 4-OH tamoxifen reduced viability in thecIgG-treated cells and, in agreement with previous reports (Argiris etal., 2004), complemented 4D5 treatment, but did not further sensitizethe cells to IFN-γ in combination with 4D5 (FIG. 48 ). The triplenegative breast cancer cell MDA-MB-231 failed to respond to mAb but didrespond to IFN-γ alone (FIG. 43 ).

erbB2 and erbB1 (EGFR) Heteromeric Kinase Activity Primarily SignalsPhenotypic Change Through the PI3-KAkt/GSK3-β Pathway and can beModified by mAb and IFN-γ or Kinase Inhibitors Such as Lapatinib.

Effects on signal transduction pathways that are activated by erbBreceptors were studied. SK-BR-3 cells were treated with the dualEGFR/erbB2 small molecule tyrosine kinase inhibitor lapatinib as well as4D5 and C225 alone and in combination to determine which signalingpathways were affected. Lapatinib treatment inactivated both the Akt andMAPK pathways as anticipated (FIG. 20A, compare lanes 5 with 6 and lanes9 with 10). Inhibiting SK-BR-3 cells with 4D5, but not C225, dominantlyinactivated Akt while not affecting MAPK activities (FIG. 20A).Combination of two distinct, receptor-specific mAbs (4D5 with C225)inactivated Akt in SK-BR-3 cells.

Disabling erbB2 in these cells was found to activate glycogen synthasekinase 3-beta (GSK3-β, which is negatively regulated by Akt-mediatedphosphorylation (Cross et al., 1995) (Lanes 5, 7, 9, and 11). Expressionpatterns of transcriptional repressors snail and slug were evaluatedbecause of the GSK3-β activation. Snail and slug proteins aredeterminants of breast cancer progression and metastases and arenegatively regulated by GSK3-β (Wu et al., 2012; Zhou et al., 2004).These experiments found that treatment with 4D5, but not C225,selectively reduced snail levels (FIG. 20A, compare lanes 5 with 6 andlanes 9 with 10 and FIG. 50 ).

Next, the response was compared to lapatinib in SK-BR-3 and MDAMB-453cells. Lapatinib treatment diminished Akt activity in both cell linesand, to a lesser extent, MAPK signaling (FIG. 20B). In addition,lapatinib induced a dose-dependent reduction in snail, but not slug.Snail levels in MDA-MB-453 cells are lower than that seen in SK-BR-3cells; nevertheless, snail is reduced upon lapatinib treatment (FIG.20B).

The PI-3K/Akt pathway was studied in greater depth using pharmacologicalinhibition. Antagonism of PI-3 kinase in SK-BR-3 cells revealed areduction in snail, but not slug (FIG. 20C). Similarly, SK-BR-3 cellswere treated with Akt1/2 inhibitor and displayed a dose-dependentdecrease in snail content (FIG. 20D). Therefore, without wishing to bebound by any scientific theory, it is concluded that erbB2 signaling inSKBR-3 operates primarily through the Akt pathway to stabilize snail andinhibition of this pathway leads to a reduction in snail protein levels.

mAb 4D5 and Interferon-γ Cooperatively Reduce Snail Protein Levels

Next, whether the combination of IFN-γ and anti-erbB2 mAb couldcooperatively reduce snail protein levels was determined. SK-BR-3 cellswere treated with cIgG or 4D5 and increasing doses of IFN-γ. IFN-γdose-dependent reduction of snail occurred only if cells were treatedwith 4D5 (FIG. 21A, compare lanes 1-5 with 6-10). IFN-γ is known toactivate GSK3-β (Beurel and Jope, 2009; Tsai et al., 2009); however,when cells are transformed by activated erbB2 signaling, IFN-γ is unableto do so. GSK3-β activation was observed to be recovered and enhanced byIFN-γ when cells were treated with mAb-erbB2. Fractionation of SK-BR-3cells revealed that snail is exclusively present in the nucleus and itsdegradation is apparent. Surprisingly, slug was found to be mostlypresent in the cytoplasm and enrichment studies reveal that slug is, infact, sensitive to these treatments (FIG. 21B). Slug content wasunaffected initially (FIG. 21A); however, by three days treatment led toreduction of Slug levels (FIG. 50 ).

In FIG. 20A, it is noted that combination of antibodies reactive witherbB2 and EGFR produced a significant reduction in snail. Therefore,combining IFN-γ with this approach was explored. Combination of 4D5 andC225 in the presence of IFN-γ produced the most dramatic reduction insnail and also reduced slug content (FIG. 21C). These results indicatethat simultaneous inhibition of heteromic kinases can also be improvedby co-treatment with IFN-γ. Therefore, whether lapatinib treatment couldbe modified by IFN-γ was examined. SK-BR-3 cells treated with increasingdoses of lapatinib and IFN-γ displayed reduced snail and slug (FIG.21D).

SK-BR-3 cells were treated with cIgG or 4D5 and increasing doses ofIFN-γ. An IFN-γ dose-dependent reduction of Snail was found only in thepresence of 4D5 (FIG. 21A, compare lanes 1-5 with 6-10). GSK3-βactivation was found to be enhanced by IFN-γ in the presence of 4D5compared to IFN-γ treated samples in the presence of control IgG. Slugcontent was unaffected initially (FIG. 21A); however, by three daystreatment led to reduction of Slug levels (FIG. 50 ).

Combination of 4D5 and C225 in the presence of IFN-γ produced the mostdramatic reduction in Snail and also reduced Slug content (FIG. 21C).Thus, these data demonstrate that erbB receptors erbB2 and EGFR,functioning as heteromeric kinases stabilize snail and slug, andcombinatorial receptor disabling in the presence of IFN-γ providesoptimal signal disruption and degradation of these proteins.

A recent study demonstrated that inclusion of IFN-β with an EGFRantibody produced a more potent anti-tumor effect than EGFR antibodyalone (Yang et al., 2014). Experiments were conducted to examine whetherIFN-β functions similarly to IFN-γ in the presence and absence of 4D5.Unexpectedly IFN-β was found to dramatically reduce snail, but not slug,even in the absence of 4D5 treatment (FIG. 22A). Experiments were alsoconducted to explore whether IFN-β effects cell viability. Surprisingly,IFN-β was found to be cytotoxic to tumor cells even at small doses inthe absence of 4D5 (FIG. 22B). Furthermore, while IFN-γ treatment ofMCF10A cells caused minimal changes to their viability, even low dosesof IFN-β were cytotoxic to these cells (FIG. 22C). In vivo studies alsodemonstrated no effects of IFN-α and anti-erbB2 mAb (Nagai et al.,Manuscript in Preparation). Therefore, without wishing to be bound byany scientific theory, it is believed that under these conditions onlyIFN-γ acts to complete phenotypic reversion engendered by anti-erbB2 mAbtherapy.

Snail Protein Degradation Occurs Through the GSK3-β Proteasomal Pathway

Snail is a labile protein that is predominantly degraded through theproteasome and, to a lesser extent, the lysosome. Proteasome degradationof snail is controlled largely by GSK3-β through phosphorylation of sixserine residues (Zhou et al., 2004). Experiments were conducted toinitially test whether treatment with IFN-γ, 4D5, or both mediated snaildegradation through the proteasome. Treatment of cells with theproteasome inhibitor MG-132 inhibited the co-treatment-mediatedreduction of snail in a dose-dependent manner, with no change in slugcontent (FIG. 23A). Conversely, inhibition of lysosomal function withchloroquine increased snail content but was unable to rescue the 4D5 andIFN-γ-mediated degradation of snail (FIG. 28 ). Without wishing to bebound by any scientific theory, it is concluded that while snail can bedegraded through the lysosome, 4D5 and IFN-γ-mediated snail degradationoccurs primarily through the proteasome. Because co-treatment favoredsnail proteasomal degradation, the role of GSK3-β was examined using thesmall molecule inhibitor CHIR99021 that is routinely used to studyGSK3-β function (Blaschke et al., 2013; Lian et al., 2012; Lian et al.,2013). Inhibition of GSK3-β in the presence of the co-treatment regimenresulted in a dose-dependent rescue of snail but led to no change inslug content (FIG. 23B).

Since treatment with 4D5 and IFN-γ activated GSK3-β and active GSK3-βcaused snail degradation, expression of a version of snail with theGSK3-β phosphorylation sites mutated to alanines would be predicted toprevent such degradation. To this end, empty vector (EV), HA-taggedsnail wild type (WT) (Kajita et al., 2004), and HA-tagged snail withserines 97, 101, 108, 112, 116, and 120 mutated to alanine (6SA) (Zhouet al., 2004) were transiently expressed in SK-BR-3 cells. The followingday, transfected cells were treated with cIgG or 4D5 in the presence orabsence of IFN-γ. After a further 24 hours, we harvested cells andseparated cytoplasmic and nuclear fractions. Both nuclear andcytoplasmic expression of exogenous WT snail was observed; however, 6SAsnail was largely present in the nucleus with only trace amounts in thecytoplasm (FIG. 23C), consistent with previous reports (Zhou et al.,2004). Further, it was found that in the nuclear fraction, exogenous WTsnail was degraded whereas 6SA snail was resistant to 4D5 andIFN-γ-induced proteasomal degradation (FIG. 23C, right panel). Analysisof total snail revealed its presence in the cytoplasmic and nuclearfractions in all samples. In cells transfected with the EV control,snail was observed in the cytoplasm only when the cells were treatedwith both 4D5 and IFN-γ (FIG. 23C, left panel) implying that bothtreatments are required for snail translocation to the cytoplasm.Collectively, these experiments identify snail as a target ofcooperative anti-erbB2 mAb plus IFN-γ treatment. This ordered therapyleads to GSK3-β-mediated proteasomal pathway degradation of the snailtranscriptional repressor. Of note, Snail knock down in SK-BR-3increased the effect of 4D5 on their proliferation (FIG. 51 ),indicating Snail decrease is important for this targeted therapy.

Kruppel Like Factor 4 (KLF4) Levels and Phenotypic Reversion Caused bymAb and IFN-γ

Disabling the erbB complex occurred through modifying the activity ofthe SHP2-PI3′ kinase and Akt pathway. Interestingly, Moral andcolleagues (Moral et al., 2009) found that KLF4 expression patterns wereincreased in tumor samples from mice with hyper-activated Akt. Enhancedexpression of KLF4 was confirmed in both dysplasias and tumors thatarose in Akt overexpressing tissues. Further analysis of human tumorsamples confirmed association between active Akt and increased KLF4expression.

The effects of ordered and combined antibody with IFN-γ were examined onKLF4 levels and Snail expression in SK-BR-3 cells stably transfectedwith GSK3-β shRNA. The ability to alter Snail functions was directlylinked to KLF4 levels and GSK3-β (FIG. 44 ). While KLF4 levels were alsodependent on GSK3-β, other signaling pathways contribute to inducechanges in KLF4 (Villarreal et al., 2010).

Effects of mAb and IFN-γ In Vivo-Combinations Reduce the Need for LargeAmounts of Targeting mAb

mAb 7.16.4 is biologically active in vivo and in vitro. In vitro mAb7.16.4 is active against cells transformed with the rat or humanerbB2/neu oncogene and disables the p185erbB2/neu kinase leading todiminished downstream p185erbB2/neu signaling (Drebin et al., 1986;Zhang et al., 1999). In several other studies, the laboratory of Dr.Mark Greene as well as others examined various optimized doses ofanti-erbB2/neu mAb therapy. 5 mg/kg of 7.16.4 mAb have been usedintravenously (Drebin et al., 1986) and/or on every other dayintraperitoneally in therapeutic studies of tumor growth (Du et al.,2013; Stagg et al., 2011).

Based on the studies above, experiments were conducted to examinewhether reduced amounts of mAb could be used if IFN-γ were providedafter therapy was initiated. As shown in FIG. 1 , when entirelysyngeneic MMTV-neu transgenic mice were treated with a sub-optimal doseof 7.16.4 (1.5 mg/kg), the mAb was unable to inhibit the growth ofH2N113 tumors. IFN-γ treatment alone also failed to significantlyinhibit tumor growth. However, the combination of suboptimal amounts of7.16.4 and IFN-γcompletely arrested the growth of H2N113 tumors.Importantly, the pattern of the data in FIG. 1 was reminiscent of the invitro tumor cell proliferation kinetics in FIG. 19B. Finally, histologicexamination of the tumor tissues after treatment revealed significantnecrosis only in mice treated with both 7.16.4 and IFN-γ (FIG. 40 ).

To examine if IFN-γ was targeted to tumor cells directly or to hostelements in the vicinity of the tumor, the expression of the IFN-γreceptor was limited on these tumor cells. Using shRNA, an IFN-γreceptor knockdown species of the H2N113 cell line was created: H2N113(IFNγR KD). The reduced expression level of IFN-γ receptor was confirmedin several distinct ways including 1) flow cytometric analysis (FIG.32A), 2) IFN-γ induced MHC expression patterns were also diminished inthese cells and finally 3) IFNγRKD tumor cells were resistant to IFN-γmediated growth suppression (FIGS. 32B and C).

IFNγRKD tumor cells were found to form progressively growing tumors, buttreatment with suboptimal mAb doses generated a modest growth reductioncomparable to that seen in the studies depicted in FIG. 1 . IFN-γ had noeffect on its own against tumors with diminished IFN-γ receptor levels.As shown in FIG. 33 , the combination effect of mAb and IFN-γ was alsoabrogated by the absence of IFN-γ receptor in the tumor cells. Withoutwishing to be bound by any scientific theory, these data indicate thatIFN-γ is required to interact directly with tumor cells to enhance theanti-tumor activity driven by the anti-p185erbB2/neu antibody andimplies that IFN-γ enhanced host responses may not be sufficientlypotent on their own to limit tumor growth. However local effects fromcells in the vicinity of the tumor that might contribute to tumorinvasion were considered.

Myeloid Derived Suppressor Cells and Foxp3+ Treg Cells

Next endogenous regulatory host processes that might be mitigated byIFN-γ were examined. Myeloid-derived suppressor cells (MDSC) are definedas CD45+ cells that are also CD11b+ and GR-1+. Co-treatment with suboptimal 7.16.4 and IFN-γ led to reduction of MDSC populations invadingtumor tissues (FIG. 45A). It was noted that there were limited butcomparable numbers of Foxp3 cells in control and treated animals. It ispossible that despite similar numbers the control Foxp3+ T cells weremore active than those that were noted in tissues of mAb and IFN-γtreated hosts. Foxp3 dependent regulatory activities has not yet beendefined since numbers of Treg cells are limited in number. However, asobserved in other studies, degradation of FOXP3 following activity ofsmall molecules that disable multiple acetyltransferases (Tip60 andp300), can alter tumor growth (Du et al., 2013). Hence both FOXP3 Tregcells and MDSC both may contribute to limitations of immune mediatedcytotoxic elimination of erbB tumors.

To illustrate the chemo-attractant effects of molecules elaborated bytumor cells on MDSC that might be affected by the combined treatment.MDSC isolated from the spleen of tumor-bearing mice were placed into theapical chamber of a transwell system. Conditioned medium from H2N113cells treated directly with mAb, IFN-γ or their combinations were testedfor their ability to attract MDSC cells to migrate into the basolateralchamber. As shown in FIG. 8D, 7.16.4 alone slightly reduced thetumor-promoted migration, but the co-treating the tumor cells themselveswith 7.16.4 and IFN-γ blocked MDSC migration. IFN-γ alone had noappreciable effects. Based on these data we suggest that immuneregulation is affected in addition to the dominant effect of mAb andIFN-γ on the tumor itself. Ordered therapy reduces regulatory cellactivity in tumor tissues.

Enhanced Role for IFN Type II Signals in erbB Cytolytic ImmuneResponses.

Studies have suggested a role for IFN-γ+CD8+ T cells (Park et al., 2010;Stagg et al., 2012; Stagg et al., 2011). Suboptimal mAb and IFN-γ wereused in syngeneic systems and effector CD8+ T cells were collected fromeach treatment group to investigate their activity against H2N113 cells.The combination therapy group showed modest but definite effector Tcells activity against p185erbB2/neu positive tumors. IFN-γ alone hadnegligible effects (FIG. 52 ).

Two types of functional macrophages have been proposed to infiltrate thetumor microenvironment and such invasion is promoted by moleculeselaborated directly or indirectly by tumor cells. Tumors were examinedfor invading anti-inflammatory, pro-tumor M2 and pro-inflammatory,cytolytic anti-tumor M1 types. mAb 7.16.4 and IFN-γ increased M1accumulation within adjacent areas of the tumor, and animals treatedwith this combination therapy had the highest M1 frequency. (FIG.41A-FIG. 41B).

In Vivo Evidence for a Role of Stem Cell Like Phenotypic Contributions.

Molecular analyses in vitro revealed that the ordered treatment ofbreast tumor cells with anti-erbB2/neu mAb and IFN-γ diminishedexpression of Snail proteins, which mediates stem cell like properties.ALDH1 expression patterns also define cancer stem cell phenotypes, andits expression correlates with breast cancer prognostic features(Douville et al., 2009; Ginestier et al., 2007). Tumors were compared atthe end of treatment for ALDH1 levels. Co-treatment with 7.16.4 andIFN-γ treatment reduced the expression of ALDH1 in p185erbB2/neu tumors.(FIG. 39 ).

Tumor cells were also examined for expression patterns of Snail. Areduced level of Snail protein was noted in tumors undergoingeradication as a consequence of mAb and IFN-γ therapy in vivo (FIG. 39). These studies directly correlate with in vitro findings described inFIG. 19A-FIG. 19D, FIG. 21A-FIG. 21D, FIG. 22A-FIG. 22C, FIG. 23A-FIG.23C, and FIG. 43A-FIG. 43D examining anchorage dependent and independenteffects of combinations of anti erbB mAb and IFN-γ.

Ordered mAb and IFN-γ Therapy can Prevent Breast Cancer Tumorigenesisand has a Combination Effect in Inhibiting Tumor Growth withChemotherapy.

To determine if these therapeutic effects were relevant to prevention oftumor development (adjuvant use of this combination) a model previouslydescribed, using small tumor inocula, was chosen for examination. Theeffects of treatment of MMTV-neu female mice were examined withcombinations of 7.16.4 and 7.9.5 mAb with or without IFN-γ, in animalsimplanted with small tumor inocula to mimic incipient tumors. A dramaticreduction was noted when IFN-γ was incorporated in the treatmentprotocol. As can be seen in FIG. 31 , this approach was able to limittumor growth in this prevention model with minimal amounts of dualtargeting mAb. These studies indicate that animals treated with orderedtherapy of mAb and IFN-γ can mount potent intrinsic cytotoxicelimination of small numbers of incipient tumor cells. These datasupport previous studies showing a delayed emergence of tumors when mAbspecific for erbB2/neu was used as an adjuvant therapy in mouseprevention studies (Finkle et al., 2004; Katsumata et al., 1995).

To extend these genotoxic observations docetaxel was added insub-therapeutic quantities to evaluate if potent tumor inhibition withsmall amounts of phenotype reversing mAb would also limit genotoxicamounts of currently employed chemotherapy (FIG. 46 ). Treatment with7.16.4 followed by IFN-γ and docetaxel led to inhibition of tumor growthcompared with other groups despite using suboptimal doses of both antierbB antibody and chemotherapy. Without wishing to be bound by anyscientific theory, optimizing phenotype reversion represents a criticalelement the evolution of precision drug therapy of breast carcinoma.

Discussion

This study demonstrates that anti-erbB2 mAb-mediated reversal of breastcancer cell malignant features permits IFN-γ mediated phenotypiceffects. These studies provide compelling evidence that mAb specific forp185erbB2/neu kinases and IFN-γ directly target erbB2+ human breastcancer cells and indicate that erbB kinase receptor complexes must bedisabled either first or simultaneously with mAb. Phenotypic reversalacts as a permissive state for the action of IFN-γ and explains many ofthe failures to properly use this cytokine in humans. IFN-γ by itself,in the absence of prior phenotypic reversal, was found to be not aseffective as a direct regulator. These studies identify orderedco-treatment as the modality that optimally affects both cell viabilityand transformation. Signaling downstream of erbB2 occurs primarilythrough the Akt pathway; and, consequently, 4D5, the precursor mAb toclinically active Herceptin, acts most profoundly on this pathway. Cellssuch as MCF-10A, which are immortalized but have not acquired malignantproperties needed to permit sustained growth in vivo, fail to beinfluenced by IFN-γ. Evidence is presented that after IFN-γ interactswith phenotype-reversed cells, it complements 4D5-mediated activation ofGSK3-β resulting in changes in snail expression.

Interferons have been reported to affect transcription in immune typecells (Qiao et al., 2013; Ucla et al., 1990); however, we find IFN-γclearly produces broad effects within the transformed breast cancercell, and this involvement in transcriptional processes relevant tophenotypic behavior may thus play a role in regulating genotoxicsensitivities. For example, IFN-γ negatively regulates skin changesassociated with UV damage by controlling the expression of severalpigmentation genes (Natarajan et al., 2014). Thus, without wishing to bebound by any scientific theory, IFN-γ may be responsible for dictatingthe response to signals that affect genomic integrity, includinggenotoxic signals caused by UV and ionizing radiation.

Prior studies implicated roles for Natural Killer cell functions in mAbtherapy (Drebin et al., 1988b) as well as other immune cell types inerbB2 tumor related processes (Park et al., 2010; Stagg et al., 2011).Although a role for host immune system contributions to anti-erbB2 mAbmediated tumor regression has been proposed, a molecular investigationof how IFN-γ elaborated by cells in the tumor microenvironment mightcooperate with kinase disabling mAbs is lacking. The studies describedherein begin to address this idea in terms of phenotype reversal andlimitation of tumorigenesis at the molecular level.

Snail is strongly implicated in the progression of breast cancer and hasbeen attributed to tumor metastasis and recurrence. Unexpectedly, whencell lines that model the various types of breast cancer were analyzed,snail expression was found to vary between different subtypes, whileslug expression was relatively constant (FIG. 29 ). In regards toerbB2/HER2+ breast cancers, this may be attributed to the known dominantactivation patterns involving Akt rather than MAPK. The data hereinunderscore the importance of ordered signals reaching the tumor and showhow failure to do so could lessen tumor eradication. The laboratory ofDr. Mark Greene demonstrated that anti-erbB2 mAb could reverse malignantphenotypes of a transformed cell (Drebin et al., 1985; Drebin et al.,1986), and now provides evidence of how IFN-γ provides a secondaryreversion of phenotype. Hence, without wishing to be bound by anyscientific theory, it is proposed that targeted therapy exists atminimum in a two-transition state, which is only accessible in adefined, linear manner; and therapeutics must be delivered in the properorder to achieve optimal results (summarized in FIG. 24 ).

Snail is perhaps best known for its role in initiating the epithelial tomesenchymal transition (EMT). While EMT is a theory currently favored bysome, it is clear from initial studies that the more differentiatedtumors respond more favorably to genotoxic injury induced by chemo- andradiation-based therapies. Therefore, complete reversion of phenotypemay be essential to achieving sensitization to more broad basedtherapeutic strategies. Designing rationalized therapies is complex andclearly requires a thorough understanding of the molecular underpinningsof the specific subtype of cancer. The results provided here wouldsuggest that activation of GSK3-β would be an appropriate approach tocomplement mAb-erbB2 based on the findings that GSK3-β inhibitionopposes efforts to degrade snail (FIG. 23A).

The findings herein represent a rational advance in the understanding ofhow to treat breast cancer from the earliest time points. Breast cancerbegins as low-grade dysplasia and ductal carcinoma in situ (DCIS), whichis defined by hyperplastic events, before progressing to palpable tumorformation. erbB2/HER2 kinase activity was detected even at the earlieststages of DCIS (Lodato et al., 1990). Anti-erbB2 mAb can be used as anadjuvant therapeutic to prevent the emergence of tumors in mice(Katsumata et al., 1995), and our data using soft agar assays (FIG. 19Cand FIG. 19D) suggests that IFN-γ would add further preventativebenefit. While clinical trials that have explored the possibility ofIFN-γ alone as a therapeutic have consistently failed, the studiesherein explain why this repeated clinical failure was seen. IFN-γadministration must be used in an ordered manner to treat alreadyphenotype reversed tumor cells in order to predictably observe enhancedtumor eradication.

Therapeutics that target p185erbB2/HER2/neu are effective forrestraining human malignant disease but are rarely curative. Disablingof the p185erbB2/neu kinase complex leads to phenotypic reversal ofmalignant properties. This phenotypic state is more sensitive togenotoxic damage by chemotherapeutic and radiation effects, or immunemediated lytic processes. These studies identified a second transitionprocess that occurs after mAb-mediated down-regulation of p185erbB2/neuproteins from the cell surface, which can be induced by IFN-γ. Thesecond transition step renders cells even more sensitive to lyticprocesses that occur in vivo by certain immune elements and tochemotherapeutics commonly used to treat breast cancer. Dramatic tumorinhibitory effects can be accomplished with minimal amounts of targetingmAb and IFN followed by chemotherapeutics (docetaxel) in vivo. Thesestudies identify a benefit of this rational approach to precisionmedicine that will be accompanied by lessened toxicity during tumortreatment.

A dramatic and almost complete arrest of tumor growth is described, evenwith suboptimal doses of anti-ErbB2/neu mAb when IFN-γ is included. Thecombination effect requires that the tumor cell itself express IFN-γreceptors. Because IFN-γ enhances antibody effects at very low doses invivo, this approach may improve targeted therapy effectiveness intissues where only low levels of antibody might penetrate.

The effect of mAb and IFN-γ are a consequence of phenotypic change onthe tumor itself. The combination effects of mAb and IFN-γ were lostwhen tumor cells with reduced IFN-γ receptor levels were targeted.Secondly, we noticed a reduction of regulatory MDSC cells that limitimmune reactions by suppressing functions of immune cells. Tumorelaborated chemo-attractants which recruit regulatory cells werediminished upon ordered treatment of tumor cells with mAb and thenIFN-γ. Studies not shown have been unable to document any comparableactivity when IFN-α or IFN-β were used (in vivo or in vitrorespectively) rather than IFN-γ. There was an increase in CD8+ T cellsable to lyse erbB2 targets (FIG. 52 ).

Foxp3+ Treg cells were detected in small numbers in the vicinity of thetumor tissues which supports previous studies that established a rolefor FOXP3 Treg in erbB tumors. In addition contributions of regulatoryMDSC cells in the local tumor environment may be important in limitingimmune elimination of breast tumors and their activity is diminishedwith ordered mAb and IFN therapy. Enhanced accumulation of M1 typemacrophages in the local tumor environment in situations using combinedmAb and IFN-γ therapy were observed. Modest enhancement of cytolytic Tcells active against erbB2 tumors was also noted. Together these datasupport an observable contributory role for the immune system intargeted therapy and a role for IFN-γ in enhancing immune eradication oferbB tumors that have undergone phenotype reversal.

mAb induced phenotypic reversion is incomplete, or at the very least, issubject to further modification, and can itself (Drebin et al., 1986;Lee et al., 2012; O'Rourke et al., 1997; Wada et al., 1990) then beacted on by IFN-γ to create a second transition state. The secondtransition involves a role of transcription factors such as Snailthought relevant to cancer stem cell features. IFN-γ alone, without thefirst reversion step is ineffective in being able to induce a malignantphenotype reversion or change in Snail levels.

In summary, this study provides a mechanistic explanation into howtargeted therapy operates at the cellular level. It shows that IFN-γprovides an extraordinary benefit to countering otherwiseoncogenic-activated signaling cascades, but only when transformed cellsare previously induced into a more normal phenotype. These findings arerelevant therapeutically because IFN-γ and the anti-erbB2 mAb(Herceptin) are FDA-approved treatments; therefore this combinationrepresents a potential benefit to patients. The studies herein indicatethat targeted therapy must be molecularly ordered to deal with distinctphenotypic states.

Experimental Procedures

Cell Culture—SK-BR-3; MDA-MB-453, MDA-MB-468, MDA-MB-231; MCF7; MCF10A;BT-474; A431; and U87 cells were obtained from the American Type CultureCollection (ATCC; Manassas, Va.). These cells were grown in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% FBS, L-glutamine (2mM), HEPES (15 mM), and antibiotics.

Chemical Compounds—Recombinant human Interferon-gamma and -beta werepurchased from BD Pharmingen and Pestka Biomedical Laboratories,respectively. Lapatinib and CHIR99021 (LC Laboratories) were resuspendedin DMSO to final concentrations of 10 mM and 50 mM, respectively. Aktinhibitor VIII (DMSO) and MG-132 (95% ethanol) (Calbiochem) wereresuspended to final concentrations of 10 mM and 20 mM, respectively.LY294002 (Cell Signaling) was resuspended in DMSO to a finalconcentration of 50 mM. Chloroquine (ddH20) and 4-OH-tamoxifen (95%ethanol) (Sigma) were resuspended to final concentrations of 50 mM and10 mM. 4D5 was kindly provided by Dr. Jeffrey Drebin. C225 was purchasedfrom Imclone (Bristol-Myers Squibb). Docetaxel was purchased from LClaboratories.

Plasmid Construction—Wild type (pcDNA3) and 6SA (pCMV-Tag 2B) snailplasmids were purchased from AddGene. The snail 6SA insert was amplifiedby PCR using primers (forward: 5′-AAAGAAGCTTATGCCGCGCTCTTTCCTC-3′ (SEQID NO: 8) and reverse: 5′-AAAGTCTAGATCAGCGGGGACATCCTGAGCAG-3′ (SEQ IDNO: 9)), which added restriction sites (HindIII and XbaI) forsub-cloning into the pcDNA3 vector. Both plasmids were sequenceverified.

Lysate Preparation and Western blotting—Cells were lysed in a RIPA-basedbuffer consisting of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 10 mM NaF, 1 mM sodiumorthovanadate, and Complete Mini Protease Inhibitors (Roche Diagnostics)on ice for 15 minutes and lysates were clarified by centrifugation(16,000×g) for 10 minutes. Supernatant was removed for analysis byWestern blot. Lysates were fractionated by SDS-PAGE using a 10%resolving gel. Gels were subsequently transferred to nitrocellulosemembranes. Membranes were blocked using 5% nonfat dried milk in 1×PBST.Membranes were washed 3 times using 1×PBST and incubated with antibodiesovernight. Protein signal was detected using Immobilon chemiluminescentsubstrate (Millipore). Blots were exposed to x-ray films.

Antibodies for western blotting—The snail, slug, pGSK3-β (S9), andtGSK3-β, pAkt (S473), tAkt, P-p44/42 pMAPK (Erk1/2) (T202/Y204), tMAPKand KLF4 antibodies were purchased from Cell Signaling and used at themanufacturer's suggested dilution. Anti-ALDH1 was purchased from Abcam.The β-actin HRP-conjugated (Sigma), HA HRP-conjugated (Roche), and Ku70(Abcam) antibodies were also used at the manufacturer's suggesteddilution. Secondary antibodies used include peroxidase-conjugated donkeyanti-rabbit and sheep anti-mouse IgG (GE Healthcare).

Lysate Preparation and Western blotting—Cells were lysed in a RIPA-basedbuffer with protease inhibitors (Roche Diagnostics) and used for Westernblot.

MTT assays—Cells were seeded in a 96-well (flat bottom) plate at1,000/well and treated the following day (Day 0). Treated cells weregrown in a 5% CO₂ incubator at 37° C. for the indicated times; and onthe day of the assay, media was exchanged for fresh media. After fourhours, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazoliumbromide) reagent was added to a final concentration of 1 mg/ml. Twohours later, lysis buffer (50% N, N-dimethyl formamide, 20% SDS, pH 4.7)was added and incubated overnight. The following day, values weredetermined by absorbance at 570 nm on a plate reader (Tecan).

Soft agar growth assays—Plates were coated with agarose mixed with DMEM(final agarose concentration of 0.8%). Cells were overlaid on the bottomlayer in an agarose (final agarose concentration of 0.2%) DMEM mixturesupplemented with the indicated therapeutic treatment. The assay wasperformed using 6-well plates and 5,000 cells per well were used. Aftertwo weeks, viable foci were visualized following overnight incubationwith MTT reagent. Each well was imaged using an Alpha Imager, and focisize was determined using NIH-endorsed ImageJ software. It wasdetermined that visible foci constituted 20 pixels using this approach;therefore, this size was used as a baseline for foci size distribution.

Transfection—Transfections were performed using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions.

Cellular Fractionation—Cells were collected in a buffer consisting of 10mM HEPES (pH 7.6), 10 mM KCl, 1.5 mM MgCl₂, 0.34M sucrose, 10% glycerol,1 mM dithiothreitol, 0.1% Nonidet P-40, 10 mM NaF, 1 mM sodiumorthovanadate, and Complete Mini Protease Inhibitors followed by a30-minute incubation on ice. Nuclei were separated by low speedcentrifugation (855×g) for 5 minutes. Supernatant containing cytoplasmicextract was removed and further centrifuged (16,000×g) for 10 minutes toremove membranes. Nuclei were resuspended in RIPA buffer (see above) andincubated on ice for 10 minutes followed by centrifugation (16,000×g)for 10 minutes.

Mouse Experiments—In these experiments, 6-10 week old female BALB/cAnNCRmice were used. mAb 7.16.4 and 7.9.5 were purified from hybridoma thatwas generated in our lab (Drebin et al., 1985; Drebin et al., 1984). Anisotype matched, anti-mammalian reovirus-3 hemagglutinin specificantibody (9BG5, mouse IgG2b) was described previously by our laboratory(Sharpe et al., 1984) and was used for control IgG. INF-γ and INF-α werepurchased from Sigma-Aldrich and R&D systems, respectively. Mice weremaintained on a standard chow diet in a barrier facility and treatedwith approval from the University of Pennsylvania IACUC in accordancewith NIH guidelines. For xenograft experiments, H2N113 cells (1×10⁶)were injected subcutaneously into both sides of the back of the mice.When tumors reached a size of 30-40 mm³ (approximately 10-12 days afterinoculation), mice were treated intraperitoneally with control (PBS),IFN-γ (three times per week), 7.16.4 (twice per week), or IFN-γ+7.16.4.Tumor sizes were monitored over the course of 7 weeks. Tumor size wasmeasured with a digital caliper and calculated using a simple algorithm(3.14×length×wide×height+6.). For in vivo treatment model, a rodenterbB2/neu transformed Balb/c breast tumor cell line, H2N113 (1×10⁶) wasinjected subcutaneously into both sides of the back of mice. Mice weredivided into four groups randomly and were treated with control IgG2a,7.16.4, IFN-γ, 7.16.4+IFN-γ. mAbs were injected 1.5 mg/kg twice a weekand IFN-γ was injected 5×10⁵ IU/kg 3 times a week after 14 days fromtumor injection. For INFα, 2.5×10⁴ IU/mouse was injected instead ofIFN-γ. Tumor size was measured with a digital caliper and calculatedusing a simple algorithm (3.14×length×wide×height+6.).

Flow cytometry—At 1 day after final treatment, spleens and tumors werecollected for single cell suspensions. Cell surface antigens werestained with the antibodies: anti-CD4, CD8, CD45, CD11b, CD11c, MHCClass II and Gr-1 (Biolegend). For intracellular staining of Foxp3 andIFN-γ, Foxp3 staining buffer set (eBioscience) was used according tomanufacturer's instruction. Cells were analyzed with FACS LSR (BDBiosciences) and FACS data were analyzed with FlowJo software (TreeStar, Ashland, Oreg.).

MDSC migration assay—To prepare conditioned medium, H2N113 cells wereseeded and cultured until sub-confluent in culture media (RPMIsupplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate and1×NEAA (Invitrogen)). Cells were then treated with 7.16.4 and/or IFN-γat indicated concentrations for 3 days then the supernatants werecollected for the migration assay. Migration of MDSC was measured by theTranswell system (pore size: 4 μm, Corning). MDSCs, which were isolatedfrom spleens of tumor-bearing mice using MACS MDSC isolation kit(Miltenyi Biotech), were placed in the upper layers. Condition media wasthen placed in the bottom layers. After 3 hr incubations in 37° C., thecells which migrated to bottom layers were collected and subjected toflow cytometry.

shRNA—GIPZ Lentivirus vecotors for GSK3β, Snail and controlnon-silencing shRNA control were obtained from GE Dharmacon. The vectorswere transfected in SK-BR-3 with Fugene 6 and selected with puromycin (1μg/ml) for stable cell line as manufactuarar's instructions. Thesilencing of target gene was confirmed by western blotting as describedabove. For IFγR1 knock down to H2N113 cells, five TRC mouse lentiviralshRNA clones targeting IFγR1 and the control pLKO.1 were obtained fromThe Open Biosystems Expression Arrest™TRC Library (Thermo Scientific).Lentiviruses were produced by VairaSafe™ Lentivirus expression system(Cell Biolabs inc., San Diego, Calif.) as manufacturer's instructions.H2N113 cells were transfected with those lentivituses and selected with1 μg/ml puromycin and FACS analysis with anti-IFγR1 antibody(Biolegend). IFγR1 knockdown was further confirmed by stimulation withIFN-γ, then analyzing their expression of MHC class I using anti-I-A/I-E(Biolegend) by FACS and proliferation.

Statistical Consideration—When applicable, statistical analysis wasperformed by Student's t-test using Microsoft Excel. At a minimum, datawith a p-value <0.05 were deemed significant. In all cases, experimentsshown are representatives that were repeated at least twice.

Histology—Tumor tissures were fixed with 10% neutral buffered formalinand embedded in paraffin. Sections were de-paraffinized and stained withH & E by the Cell Imaging Core of the Abramson Cancer ResearchInstitute.

Flow cytometry—At 1 day after final treatment, spleens and tumors werecollected for single cell suspensions. Tumors tissue was cut anddigested with Collagenase P for 1 hr, then dispase (Stem Cell) and DNase(1 μg/ml, Roche) were added and incubated for 5 minutes. Then cells arefiltrated by cell strainer (Falcon). Single suspension of tumor tissuecells were stained with CD45, F4/80, CD11b, and CD206 antibodies(Biolegend). Cells were analyzed with FACS LSR (BD Biosciences) and FACSdata were analyzed with FlowJo software (Tree Star, Ashland, Oreg.).

In vitro proliferation assays—10⁴ H2N113 transfected with empty vectorand IF□R1 shRNA vector were cultured in 96-well plates for 5 days withindicated concentration of IFN-γ. Relative cell numbers were measured byLDH activities of total cell lysates by CytoTox 96® cytotoxicity assaykit (Promega). The means of each data set were analyzed using Student'st-test with a two tailed distribution.

Cytotoxicity assay—CD8+ T cells (effector cells) were collected fromspleen cells and sorted as CD8+CD3+ cells by FACS Aria II (BDBiioscience). H2N113 cells (target cells) were seeded in the 96-wellplate at 10,000/well and incubated with CD8+ T cells at the ratio of1:20. After overnight incubation, the plate was centrifuged and 50 μl ofsupernatants were measured for LDH release using the CytoTox 96®cytotoxicity assay kit (Promega). LDH release (%) was calculated as[A]sample−[A]minimum/[A]max−[A]minimum×100%, where [A]max is theabsorbance value of a positive control (Triton X-100) in which completetarget cell lysis occurred and [A] minimum is negative control (withouteffector cells).

Example 4

IFN-γ Enhances Activity of Dual Antibody

Previous results, with two anti-p185erbB2/neu antibodies 7.16.4 and7.9.5 mAbs, showed that dual antibody therapy targeting distinctepitopes of the p185 ectodomain (Drebin et al., 1988) is far moreeffective therapeutically in treating established tumors and thisapproach has been now clinically adopted with the use of trastuzumabplus pertuzumab for breast cancers. Using H2N113 mammary tumorsimplanted in MMTVneu mice (Stagg et al., 2011; Du et al., 2013),experiments were performed to evaluate whether IFN-γ could improve theeffects of dual anti-p185 mAbs. 1 million H2N113 cells were injectedinto MMTVneu mice subcutaneously. Once tumors reached 30-50 mm³, micewere divided into four groups for different treatments. After two weeksof treatments (7.16.4 30 μg/mouse, 7.9.5 100 μg/mouse, twice per week;IFN-γ, 10⁴ IU/mouse). Spleen cells were isolated and studied. As shownin FIG. 30A-FIG. 30B, IFN-γ improved therapeutic activity oftwo-antibody as determined by the CD8+ dendritic cell (DC) populationsin spleen and cytotoxicity CD8+ T cells from spleen against tumor cells.

This regimen was then extended in a “preventative” model, in whichtreatment started within one day of inoculation of small tumor inocula(0.25 million H2N113 cells) to mimic a preventative therapeuticsituation in which very small tumor nests remain after therapeuticsurgery (Greenberg and Greene, 1976). Treatments started one day afterinoculation of tumor cells. As shown in FIG. 31 , more than half ofinocula became palpable in the control group on day 16. Treatment with 2antibodies prevented tumor appearance in half of injections until day25. At the end of the experiment (day 32), half of inocula in 2antibodies treated group were still free of palpable tumors. Addition ofIFN-γ to the 2 antibodies further prevent the appearance of palpabletumors and near 69.8% of inocula remain free of palpable tumors at theend of the experiment.

Combination Activity of IFN-γ is Dependent on the IFN-γ ReceptorExpressed on Tumor Cells.

To examine if IFN-γ was targeted to tumor cells directly the expressionof the IFN-γ receptor was limited on these tumor cells. Using shRNA, anIFN-γ receptor knockdown species of the H2N113 cell line was created:H2N113 (IFNγR KD). The same suboptimal mAb approach was employed tomaximize the demonstration of the combined role of these two reagents.The reduced expression level of IFN-γ receptor was confirmed by flowcytometric analysis (FIG. 32A-FIG. 32C). IFN-γ induced MHC expressionwas diminished in these cells and tumor cells were resistant to IFN-γmediated growth suppression (FIG. 32B and FIG. 32C).

IFN-γ receptor low expressing tumor cells form progressively growingtumors. Treatment with suboptimal mAb 7.16.4 doses generated a modestgrowth reduction. IFN-γ had no effect on its own against tumors withdiminished IFN-γ receptor levels. As shown in FIG. 1 and FIG. 33 , thecombination effect of mAb and IFN-γ was also abrogated by the absence ofIFN-γ receptor in the tumor cells. Without wishing to be bound by anyscientific theory, these data indicate that IFN-γ is required tointeract directly with tumor cells to enhance the anti-tumor activitydriven by the anti-p185erbB2/neu antibody and implies that IFN-γenhanced host responses may not be sufficiently potent on their own tolimit tumor growth.

IFN-γ Enhances the Activity of Anti-p185 Antibody in the Presence ofChemotherapeutic Agents.

In current clinical treatment for HER2 positive tumors, anti-HER2antibody trastuzumab is administrated together with chemotherapeuticagents, such as docetaxel. To examine if IFN-γ further enhances theactivity of antibody in the presence of chemotherapies, H2N113 cellswere implanted into MMTVneu mice and subjected mice to controltreatment, 7.16.4+docetaxel, or 7.16.4+docetaxel+IFN-γ after tumorsbecame palpable (20-50 mm³). As shown in FIG. 34 , 7.16.4 and docetaxelreduced the tumor growth. When IFN-γ was added to the treatment, tumorgrowth was further reduced and the average tumor size in the tripletreatment group even showed trend of shrinking of tumors.

Benefit of Anti-PD1 Antibody to IFN-γ Included Treatments.

Previous reports (Stagg et al., 2011) as well as unpublished study inour lab indicated that IFN-γ treatment could induce the expression ofPD-L1 on the tumor cells. The implanted tumors (H2N113) in the syngeneicMMTVneu mice model, were used to test whether anti-PD1 antibody couldprovide benefit by blocking PD-L1 and PD1 interaction and boosting hostimmunity against tumors. As shown in FIG. 35 , treatment of anti-PD1antibody together with anti-p185 mAb 7.16.4 and IFN-γ showed betteractivity than treatment with only 7.16.4 and IFN-γ.

New Data on the IFN Fusion Protein

A Mouse Version of the Fusion Protein 4D5scFvZZ-mIFN-γ

The present invention provides a fusion construct 4D5scFvZZ-IFN-γ (SEQID NO: 2), which contains the human IFN-γ sequence. While the constructwas designed for use in humans, a similar construct 4D5scFvZZ-mIFN-γ wasconstructed to contain the mouse IFN-γ sequence to have a betterassessment for the in vivo activity in the mouse model currently beingtested. Athymic nude mice were inoculated with 5×10⁵ T6-17 cells. Micecarrying tumor were treated with 4D5scFvZZ-mIFN-γ or the clinically usedtrastuzumab (4D5) at a very low dose of 0.125 mg/kg, five times per weekvia i.p. injection. As shown in FIG. 36 , 4D5scFvZZ-mIFN-γ has muchbetter activity than the anti-HER2 antibody 4D5 to limit the growth ofT6-17 tumors.

The Activity of 4D5scFvZZ-mIFN-γ is Also Dependent on the IFN-γ Receptorin the Tumor Cells

To examine if IFN-γ receptor is required for the activity of therecombinant fusion protein, a shRNA knock-down of the IFN-γ receptor inT6-17 was performed. T6-17 cells were transfected with either the vectoror the IFN-γ receptor shRNA to establish T6-17 (Vector) and T6-17(IFN-γR KD) cell lines respectively. An in vivo experiment was performedas in FIG. 36 . As shown in FIG. 37A-FIG. 37B, while the activity of4D5scFvZZ-mIFN-γ is clear on the control cell line T6-17 (Vector), itsactivity against the T6-17 (IFN-γR KD) was diminished.

Example 5

Ordered Combination of erbB Targeted Antibody and Immune Therapy ofBreast Cancer

Summary: Reversion of the malignant phenotype of erbB2-transformed cellscan be driven by monoclonal antibodies (mAb) that bind the p185erbB2/neuectodomain (anti-erbB2/neu mAb) and disrupt the transforming kinaseactivity. Cells treated with disabling mAb have limitedanchorage-independent and -dependent growth capacity in vitro, anddiminished tumor growth in vivo. This study examined the effects ofinterferon-γ alone on erbB2-positive cells and those that were alsotreated with anti-erbB2/neu mAb. Interferon-γ was without effect on itsown, indicating that immune therapies mediated by this cytokine aloneare unlikely to be beneficial. However, it was discovered that treatmentof the tumors with anti-erbB2/neu mAb initially for at least 24 hoursprior to IFN-γ or concomitant with IFN-γ led to dramatic inhibition oftumor growth in vivo. The tumor growth inhibition could be accomplishedwith “minimal amounts of mAb” and reflect a combination effect of thetwo modalities. The use of IFN-γ in conjunction with mAb revealed thatthe IFN-γ effect was mediated on the tumor itself since dual treatmentof IFN-γR null tumors did not show combination effects. IFN-γ hadmoderate effects on host cell constituents. An increase of M1 macrophageaccumulation was noted in the tissues as well as diminished myeloidderived suppressor cells. The tumor cells treated with mAb and IFN-γunderwent changes in phenotypic markers reflecting loss of stemcell-like properties, while mAb treatment alone did not accomplish thisphenotypic change.

Recognition that mAb could disable the p185erbB2/HER2/neu tyrosinekinase receptor complex and also lead to reversal of the malignantphenotype challenged the dogma that transformed cells could onlyprogressively become more abnormal (Schechter et al. (1984); Drebin etal. (1985). Reversal of the malignant phenotype by anti-erbB2 mAb occursrapidly beginning within 24 hours (Drebin et al. (1986); Lee et al.(2012); O'Rourke et al. (1997); (Qian et al. (1994)) beginning with downregulation of p185erbB2/neu receptor tyrosine kinase proteins anddiminished enzymatic activity (Drebin et al. (1986); Furuuchi et al.(2007); Sliwkowski et al. (2013); Zhang et al. (2007); Drebin et al.(1988); Wada et al. (1990)). erbB2/HER2/neu genes are amplified and theprotein overexpressed in ˜30% of breast cancer patients and thisoncogenic alteration is associated with aggressive disease, increasedrecurrence rate, and reduced patient survival. All of these clinicalfeatures are improved by anti-ErbB2 mAb therapy (Kiessling et al.(2002); Seidman et al. (2001); Romond et al. (2005); Baselga et al.(2001); Hudis et al. (2007); Meric-Bernstam et al. (2006)).

Both a contributory role of Natural Killer (NK)-like cells inantierbB2/HER2/neu mAb in vivo therapy (Clynes et al. (2000); Park etal. (2010); Junttila et al. (2010); Hurvitz et al. (2012); Drebin et al.(1988)) as well as effects from adaptive immunity in certain stages oftumors subjected to anti-erbB2/neu mAb therapy have been identified.Adaptive responses include CD8+ T cells19 as well as CD4+ cells(Mortenson et al. (2013)). Stagg et al. suggested that mAb therapyrequires type 1 and 2 interferons (IFN), and found IFN-γ induced CD8+ Tcells were determinants for effective tumor inhibition (Stagg et al.(2011)). IFN-γ, a cytokine that plays diverse roles in innate andadaptive immune response, was one of the first recombinant proteinsexamined in human cancer therapy (Platanais et al. (2013); Bekisz et al.(2013); Balachandran et al. (2013); Ernstoff et al. 1987). RecombinantIFN-γ exhibits antiproliferative and apoptotic effects (Zaidi et al.(2011)); however, despite these characteristics, the clinical use ofIFN-γ alone for cancer therapy in humans is ineffective (Zaidi et al.(2011); Miller et al. (2009)) and has been all but abandoned for thatuse.

mAb 7.16.4 is biologically active against cells transformed with the rator human erbB2/neu oncogene and disables the p185erbB2/neu kinaseleading to diminished downstream p185erbB2/neu signaling (Drebin et al.(1985); Zhang et al. (1999)). Previous studies examined variousoptimized doses of anti-erbB2/neu mAb therapy. 5 mg/kg of 7.16.4 mAbhave been used intravenously (Drebin et al. (1985)) and/or on everyother day intraperitoneally in therapeutic studies of tumor growth(Stagg et al. (2011); Du et al. (2013)). This study examined if reducedamounts of mAb could be used if IFN-γ were provided after therapy wasinitiated. As shown in FIG. 1 , 7.16.4, anti-p185erbB2/neu mAb, whengiven at a sub-optimal dose (1.5 mg/kg), was unable to inhibit thegrowth of H2N113 tumor in entirely syngeneic MMTV-neu transgenic mice.IFN-γ treatment alone also failed to cause significant inhibition of thegrowth of the tumor. However, the combination of suboptimal amounts of7.16.4 and IFN-γ “completely arrested” the growth of H2N113 tumors.Histological examination of the tumor tissues after treatment revealedsignificant necrosis only in mice treated with both 7.16.4 and IFN-γ(FIG. 40 ).

To examine if IFN-γ was targeted to tumor cells directly limited theexpression of the IFN-γ receptor was limited on these tumor cells. UsingshRNA, an IFN-γ receptor knockdown species of the H2N113 cell line wascreated: H2N113 (IFN-γR KD). The same suboptimal mAb approach wasemployed to maximize the demonstration of the combined role of these tworeagents. The reduced expression level of IFN-γ receptor was confirmedby 1) flow cytometric analysis (FIG. 32A), 2) IFN-γ induced MHCexpression was diminished in these cells and 3) tumor cells wereresistant to IFN-γ mediated growth suppression (FIG. 32B and FIG. 32C).

IFN-γ receptor low expressing tumor cells form progressively growingtumors. Treatment with suboptimal mAb doses generated a modest growthreduction comparable to that seen in the studies depicted in FIG. 1 .IFN-γ had no effect on its own against tumors with diminished IFN-γreceptor levels. As shown in FIG. 33 , the combination effect of mAb andIFN-γ was also abrogated by the absence of IFN-γ receptor in the tumorcells. These data indicate that IFN-γ is required to interact directlywith tumor cells to enhance the anti-tumor activity driven by theanti-p185erbB2/neu antibody and implies that IFN-γ enhanced hostresponses may not be sufficiently potent on their own to limit tumorgrowth.

There is a growing momentum for immune therapy of tumors. Even thoughsignificant effects of IFN-γ by itself has not been seen, endogenousregulatory host processes that might be mitigated by IFN-γ wereexamined. Myeloid-derived suppressor cells (MDSC) are defined as CD45+cells that are also CD11b+ and GR-1+. Cells with the MDSC phenotype fromtumors of treated or control BALB/c mice were identified. Co-treatmentwith sub optimal 7.16.4 and IFN-γ led to reduction of MDSC populationsinvading tumor tissues (FIG. 38A).

In vitro cell migration studies were performed to illustrate thechemo-attractant effects of molecules elaborated by tumor cells thatmight be affected by the combined treatment. MDSC isolated from micespleens were placed into the upper chamber. As shown in FIG. 38B,conditioned medium from H2N113 cells treated directly with mAb, IFN-γ ortheir combinations attracted MDSC cells to migrate into the lowerchamber. 7.16.4 alone slightly reduced the tumor-promoted migration, butthe co-treatment of 7.16.4 and IFN-γ of the tumor cells themselvesblocked MDSC migration, while IFN alone had no appreciable effects.

Two types of functional macrophages have been proposed to infiltrate thetumor microenvironment also promoted by molecules elaborated directly orindirectly by tumor cells. Tumors were examined for invadinganti-inflammatory, pro-tumor M2 and pro-inflammatory, anti-tumor M1types. MAb7.16.4 and IFN-γ increased M1 accumulation within adjacentareas of the tumor, and animals treated with this combination therapyhad the highest M1 frequency. (FIG. 41A-FIG. 41C).

Studies have suggested a role for IFN-γ induced CD8+ T cells in certainphases of the anti-erbB2/neu mAb treatment in some tumor model systems(Stagg et al. (2011); Stagg et al. (2012)). Sub-optimal mAb and IFN-γwere used in syngeneic systems and effector T cells were collected fromeach treatment group to investigate their activity against H2N113 cells.The combination therapy group showed only modest effector T cellsactivity against p185erbB2/neu positive tumors. IFN-γ alone had minimaleffects (not shown).

Molecular analyses revealed that in vitro the ordered treatment ofbreast tumor cells with anti-erbB2/neu mAb followed by IFN-γ, but notwith IFN-γ followed by anti-erbB2/neu mAb, diminishes expression ofsnail proteins, which mediates stem cell like properties (Runkle et al2014 submitted). ALDH1 expression patterns also define cancer stem cellphenotypes, and its expression correlates with breast cancer prognosticfeatures (Douville et al. (2009); Ginestier et al. (2007)). Tumors werecompared at the end of treatment for ALDH1 levels. Co-treatment with7.16.4 and IFN-γ treatment reduced the expression of ALDH1 inp185erbB2/neu tumors (FIG. 39 ).

Discussion

Therapeutics that target p185erbB2/HER2/neu are effective forrestraining human malignant disease but are rarely curative. Disablingof the p185erbB2/neu kinase complex leads to phenotypic reversal ofmalignant properties. This phenotypic state is more sensitive togenotoxic damage by chemotherapeutic and radiation effects, or immunemediated lytic processes. The studies herein identified a secondtransition process that occurs after mAb-mediated down-regulation ofp185erbB2/neu proteins from the cell surface, which can be induced byIFN-γ. The second transition step renders cells even more sensitive tolytic processes that occur in vivo by certain immune elements.

This study describes a dramatic and complete arrest of tumor growth evenwith suboptimal doses of anti-ErbB2/neu mAb when IFN-γ is included.Moreover, the combined effect requires that the tumor cell itselfexpress IFN-γ receptors. Because IFN-γ enhances antibody effects at verylow doses in vivo, it would improve targeted therapy effectiveness intissues where only low levels of antibody might penetrate.

The effect of mAb and IFN-γ appears to be a consequence of phenotypicchange on the tumor itself. The combination effects of mAB and IFN-γwere lost when tumor cells with reduced IFN-γ receptor levels weretargeted. Secondly, a reduction of regulatory MDSC cells that limitimmune reactions by suppressing functions of immune cells was noticed.Tumor elaborated chemo-attractants which recruit regulatory cells werediminished upon ordered treatment of tumor cells with mAb and thenIFN-γ. Studies not shown in this Example were unable to document anycomparable activity when IFN-α or IFN-β were used (in vivo or in vitrorespectively) rather than IFN-γ. (not shown).

An accumulation of M1 type macrophages in the local tumor environment insituations using combined mAb and IFN-γ therapy was also observed.Together these data support a role for the immune system in targetedtherapy and a role for IFN-γ in enhancing immune eradication of erbBtumors that have undergone phenotype reversal. However immunemodifications induced by IFN-γ alone are ineffective on their own.

mAb induced phenotypic reversion is incomplete, or at the very least, issubject to further modification, and can itself (Drebin et al. (1986);Lee et al. (2012); O'Rourke et al. (1997); Wada et al. (1990)) then beacted on by IFN-γ to create a second transition state. The secondtransition involves a role of transcription factors thought relevant tocancer stem cell features (Example 3).

IFN-γ alone, without the first reversion step is ineffective in beingable to induce a malignant phenotype reversion.

ALDH1 is diminished in tumor cells treated in vivo with combined mAb andIFN therapy. Elevation of ALDH1 enzyme levels correspond to erlotinibresistance in erbB1 (EGFR) mutated human tumors (Corominas-Faja et al.(2013)). Thus metabolic changes occur within tumor cells with stem celllike properties, that are amenable to manipulation by phenotypicreversion best promoted by anti-p185erbB2/neu mAb and by IFN-γ.Consequently, tumor tissues should be examined diagnostically forexpression of IFN-γ receptor and aldolase in situations in whichtargeted therapy might be administered.

Combining targeted therapy with immune therapy in an ordered way maylead to a greatly reduced need for the mAb components and possibly thegenotoxic molecules needed to treat humans.

Supplemental Materials and Methods

Histology

Tumor tissures were fixed with 10% neutral buffered formalin andembedded in paraffin. Sections were de-paraffinized and stained with H &E by the Cell Imaging Core of the Abramson Cancer Research Institute.

Flow Cytometry

At 1 day after final treatment, spleens and tumors were collected forsingle cell suspensions. Tumors tissue was cut and digested withCollagenase P for 1 hr, then dispase (Stem Cell) and DNase (1 μg/ml,Roche) were added and incubated for 5 minutes. Then cells are filtratedby cell strainer (Falcon). Single suspension of tumor tissue cells werestained with CD45, F4/80, CD11b, and CD206 antibodies (Biolegend). Cellswere analyzed with FACS LSR (BD Biosciences) and FACS data were analyzedwith FlowJo software (Tree Star, Ashland, Oreg.).

In Vitro Proliferation Assays

10⁴ H2N113 transfected with empty vector and IFγR1 shRNA vector werecultured in 96-well plates for 5 days with indicated concentration ofIFNγ. Relative cell numbers were measured by LDH activities of totalcell lysates by CytoTox 96® cytotoxicity assay kit (Promega). The meansof each data set were analyzed using Student's t-test with a two taileddistribution.

Example 6 Summary

Efforts of Dr. Mark Greene's laboratory have focused on improvingrationally designed and developed targeted adjuvant therapy for humanbreast cancer to prevent reoccurrence and limit metastatic spread. Thenew therapeutics appear to be useful for treating advanced erbBresistant breast cancers.

Evolution of the therapeutic approach described herein has been theresult of success in developing completely novel erbB targetingcompounds coupled with enhanced molecular understanding of how todisable erbB transformation of breast cancer in vivo. A new therapeuticfor use in human breast cancer has been developed, and is expected enterthe clinic in the near future.

The therapeutic was developed to combine features of targeted therapyand immune therapy. These two therapeutic approaches can work togetherin an unexpected way. Two forms of this second therapeutic species havebeen developed. One approach simply uses two approved molecules used ina specific sequence. The second form is a new single molecule builtthrough genetic engineering that combines the two proteins together.Studies of the two molecules used in a defined sequence have found thata Herceptin-like antibody protein already used to treat cancer in humansbecomes far more efficacious when followed sequentially by an immunerecombinant protein, interferon-gamma. Interferon-gamma is also approvedfor human use but not for breast cancer. This sequential use of targetedand immune therapies is expected to be able to enter the clinic in thenear future. Only ⅓ the amount of the Herceptin like antibody was foundto be required to achieve even more complete tumor eradication whensequential treatment with interferon-gamma was added to the regimen. Inaddition, new antibody forms were created that combine theHerceptin-like antibody properties with the Interferon in a single newrecombinant protein to treat human breast cancer.

Progress Studies relating to new recombinant proteins that disable themalignant phenotype of breast cancer cells are pursued. Theaccomplishments as they relate to individual goals are summarized below.

Aim: In this aim disabling the p185erbB2/HER2/neu receptor withmonoclonal antibodies followed by recombinant interferon proteins isstudied. There is also a focus on studies of the humanized pan erbBrecombinant species and scFv forms of Herceptin antibodies linked toIFN-γ as a recombinant structure.

The pan erbB mAb-like S22-23-Fc loop body species, modeled from theectodomain of erbB2 and capable of limiting erbB2 and erbB3 activity inhuman breast cancer, has been created. Recombinant technology is alsoused to embed the IFN-γ sequence to the carboxy termini of a new mAb(Herceptin) scFv species. These erbB receptor disabling moleculescoupled with IFN-γ can dramatically limit growth of erbB2 transformedhuman breast cancer tumors in vivo in animal models.

Several studies were conducted that indicate paired disabling of erbBreceptors by mAb along with IFN-γ signals can optimally reverse themalignant cell phenotype in vitro and in vivo. Studies have alsoidentified certain of the molecular mechanisms by which targeted therapycan be augmented by recombinant IFN-γ. The mechanisms of combiningoncoprotein targeting effects and the tumor effects of IFN-γ are alsobeing studied. These studies will be important as it has been discoveredthat combining targeted therapy and IFN-γ reduces tumor growth morecompletely, reduces the need for targeted antibodies by 66% and reducesamounts of genotoxic molecules (paclitaxel, docetaxel) needed to causetumor death by 50%.

Specific Progress to Date.

Combination effect of targeted and immune therapy using recombinantproteins. Features of the ordered combination of targeting mAb followedsequentially with recombinant IFN-γ. In addition, significant progresshas been made with the recombinant scFv 4D5-human Fc linked to IFN-γ andthe pan erbB recombinant S22-human Fc form coupled with IFN-γ on humanbreast tumor cell growth in vitro and in vivo. Certain of theseconstructs are designed to express recombinant 4D5 and release IFN-γupon binding because of a labile cleavage site embedded in theconstruct.

The data are striking, and the creation of an engineered scFv-specieslinked with IFN-γ at the carboxy terminus shown in FIG. 36 is moreeffective than 4D5 (the Herceptin) at limiting erbB2 amplifiedtransformed cell growth. 4D5scFvZZ-mIFN-γ has better activity than 4D5in the in vivo study. One day after injection of T6-17 cells s.c. intoathymic mice, mice were randomly grouped and treated with control, 4D5or 4D5scFvZZ-mIFN-γ at the dosage of 0.125 mg/kg, 5 days per week, viai.p. injection. Mice treated with 4D5scFvZZ-mIFN-γ had verysignificantly smaller tumors.

Experiments that were conducted also examined the activity of the S22-Fcpan erbB inhibitory molecule alone and in combination with IFN-γ. Thesestudies have revealed that even this unique pan erbB inhibitor can bemade functionally more effective by ordered therapy with IFN-γ (Notshown).

Targeted Therapy with Anti-erbB2 mAb and Sequential IFN-γ Reduces theAmount of Antibody Needed for Phenotypic Reversal.

The possibility of reducing the amount of targeting mAb administeredwhen its effect was enhanced with sequential IFN-γ was examined.

Optimized doses of anti-erbB2/neu mAb therapy have been determined andtypically 5 mg/kg of 7.16.4 mAb has been used intraperitoneally 2-3times/week in therapeutic studies of tumor growth. Experiments wereconducted to examine if reduced amounts of mAb could be used if IFN-γwere provided after therapy was initiated. As shown in FIG. 1 , 7.16.4anti-p185erbB2/neu mAb, when given at a sub-optimal dose of 1.5 mg/kg (⅓of the normal dose) was unable to inhibit the growth of H2N113 tumorsignificantly in entirely syngeneic MMTV-neu transgenic mice. IFN-γtreatment alone also failed to cause significant inhibition of thegrowth of the tumor. However, the combination of low dose 7.16.4 andIFN-γ “completely arrested” the growth of H2N113 tumors. Histologicalexamination of the tumor tissues after treatment revealed significantnecrosis only in mice treated with both 7.16.4 and IFN-γ (not shown).

The Locus of Action of IFN-γ Appears to be Dominantly the Tumor Itself.

To examine if the role of IFN-γ was targeted to tumor cells directly,the expression of the IFN-γ receptor was limited on these tumor cells.Using shRNA, an IFN-γ receptor knockdown version of the H2N113 cell linewas created: H2N113 (IFNγR KD) (FIG. 33 ). The same suboptimal mAbapproach was employed to maximize the demonstration of the combined roleof these two reagents. The reduced expression level of IFN-γ receptorwas confirmed by 1) flow cytometric analysis, 2) that IFN-γ induced MHCexpression was diminished in these cells.

As shown in FIG. 33 IFN-γ receptor low expressing tumor cells led toprogressively growing tumors. Treatment with suboptimal mAb dosesgenerated a modest growth reduction comparable to that seen in thestudies depicted in FIG. 1 . IFN-γ had no effect on its own againsttumors with diminished IFN-γ receptor levels. As shown in FIG. 33 , thecombination effect of mAb and IFN-γ was also abrogated by the absence ofIFN-γ receptor in the tumor cells. These data indicate that IFN-γ isrequired to interact directly with tumor cells to enhance the anti-tumoractivity driven by the anti-p185erbB2/neu antibody and indicate thatIFN-γ alone enhanced host response is not sufficiently potent to limittumor growth.

Implications of Reduced Amounts of Targeting Antibody and CytotoxicReagents.

Therapeutics that target p185erbB2/HER2/neu are effective forrestraining human malignant disease but are rarely curative.Down-modulation of the receptor from the cell surface, leads tophenotypic reversal of malignancy. This phenotypic state is moresensitive to genotoxic damage by chemotherapeutic and radiation effects,or immune mediated lytic processes. Studies described herein identifieda second transition that can be induced by IFN-γ. The second transitionstep renders cells more sensitive to lytic processes that occur in vivoby immune elements or cytotoxic chemotherapeutics as shown in FIG. 42 .

These studies indicate a dramatic and complete arrest of tumor growtheven with suboptimal doses of anti-erbB2/neu mAb when IFN-γ is included.Moreover, the combined effect requires that the tumor cell itselfexpress IFN-γ receptors. These studies indicate the importance of thecombination because IFN-γ enhances antibody effects at very low doses invivo thereby improving targeted therapy effectiveness in tissues whereonly low levels of antibody might penetrate. In studies not shown inthis example been unable to document any comparable activity when IFN-αor IFN-β were used (in vivo or in vitro respectively) rather than IFN-γ.

Additionally, an accumulation of M1 type macrophages was observed in thelocal tumor environment in situations using combined mAb and IFN-γtherapy. Together, these data support a modest role for the immunesystem in targeted therapy and a role for IFN-γ in enhancing immuneeradication of erbB tumors that have undergone phenotype reversal.Importantly, it was discovered that immune modifications induced byIFN-γ alone are ineffective, indicating a more fundamental process ofphenotypic transitions is responsible for the synergistic activities.

Lay Abstract

A new therapeutic approach was studied to treat and eradicate advancedbreast cancer and also to preempt tumor re-emergence after breast cancersurgery.

A family of antibody-like proteins have been produced using recombinantengineering. These proteins are pan erbB inhibitors that bind anddisable erbB2 active receptors and erbB2-erbB3 receptor forms. Some ofthese recombinant proteins have been engineered to carryInterferon-gamma, an immune activating molecule. One family of therecombinant proteins has shown promise in limiting growth of humanbreast tumors that accumulate more than one kind of mutation in theirgenes and become resistant to current therapy. The recombinant proteins,which combine erbB inhibition with an immune activator such asinterferon-gamma, are particularly effective at reversing growthproperties of doubly transformed tumor lines, that is breast tumorswhich have more than one mutated gene, in preclinical studies.

Another major set of studies is using both the synthetic molecules andengineered antibody molecules to prevent tumor formation and tumorreoccurrence. Initial studies have been made in a preclinical setting toshow that one anti-HER2 antibody when concomitantly used withInterferon-gamma can actually limit the development of breast tumors ina small animal model. Those studies are extended to see if it ispossible more completely arrest tumor development using the syntheticmolecules or two antibodies that bind to erbB receptors alone and incombination with immune activators like Interferon-gamma. A unique smallanimal model is be used to determine if these therapeutics can actuallyprevent the hyperplasia that occurs very early in breast cancerdevelopment and then prevent tumor formation completely. Both of the newtherapeutics are expected to reach the clinic in the near future.

Additional Studies: The studies include biochemical and mechanisticstudies. These are be extended to in vivo studies using a uniquetransgenic breast cancer model (MMTV NeuT-tdTomato) that allows cancercells to be followed because a tomato red chromophore is incorporated inthe oncogenic neu construct.

Prevention of hyperplasia and tumorigenesis by combination effects ofmAb and IFN-γ in transgenic models.

A). Prevention of tumor occurrence is studied. MMTV-neu BALB/ctransgenic mice have been developed by the laboratory of Dr. Mark Greeneto study the development of incipient erbB2/HER2/neu mammary tumors infemale mice. In the MMTV-neu model, rat oncogenic neu is expressed underthe control of the MMTV promotor and tumors arise ˜30 weeksstochastically in mice that have delivered and nursed a litter. Allcontrol females develop tumors by ˜60 weeks of age. In vivoadministration of mAb directed at the ectodomain of p185erbB2/neu inMMTV-neu transgenics prior to mammary tumor development significantlyinhibits the pre-neoplastic state associated with the initial events oftumorigenesis and delays tumor onset and final extent of metastases.

B). Dual antibody therapy targeting distinct epitopes of the p185ectodomain is far more effective therapeutically in treating establishedtumors. Experiments are conducted to evaluate if the effects of dualmAbs can be further improved in prevention models of breast tumors. Theexperiments examine whether IFN-γ could augment the preventative potencyof dual mAb in a simple model of prevention of breast tumor.

Two anti-p185erbB2/neu antibodies, mAbs 7.16.4 and 7.9.5, bind todistinct epitopes on the p185 ectodomain. A “preventative” preliminarystudy has been completed. In this model inoculation of small tumormasses is used to mimic a preventative therapeutic situation in whichvery small tumor nests remain after therapeutic surgery. In thispreliminary study, 9 out of 10 tumors became palpable in the controlgroup by day 10. Treatment with 2 antibodies prevented tumor appearancein 3 out of 10 mice during the first two weeks. When the two-antibodyregimen was combined with IFN-γ most (8 out of 10) in this group failedto develop tumors. Although some tumors became palpable later in thecourse of the experiment, 60% of the 2 antibodies plus IFN-γ groupremained tumor free. This extraordinary observation is extended.

C). Prevention studies are extended to a transgenic model recentlydeveloped using MMTV NeuT-tdTomato red constructs. Tumors developstochastically in females and this genetic construct permitsvisualization of the earliest lesions (hyperplasia) and the spread ofneoplastic tumors. Thus, this novel transgenic allows the visualizationof the tumor as it arises stochastically in the breast and metastasizes.Tumors arise at 23-30 weeks and all female mice have tumors by 50 weeks.

These studies examine an effect of mAb antibodies and IFN-γ on theearliest lesions of breast cancer tumor development, when administeredprior to tumorigenesis. Experiments are conducted to examine if theseanimals which develop sequential changes in the breast includinghyperplasia and then neoplasia is prevented from developing either ofthese lesions. The power of the model is that effects of early spread oftumor from the breast can be examined. In these experiments twoantibodies plus IFN-γ is administered twice weekly beginning at week 12after birth. Another group of female mice will receive two antibodiesalone or IFN-γ alone and breast tumors and metastases spread monitored.These studies indicate that the combination effects of mAb and IFN-γlimit tumor occurrence and metastatic spread as preliminary studiesindicate.

D). Studies also extend to the use of 2 targeted antibodies, concurrentor sequential administration of IFN-γ, followed by small doses ofdocetaxel in pre-clinical models of prevention.

Example 7

Sequential Disabling of the erbB Pathway Followed by IFN-γ ModifiesPhenotype and Enhances Genotoxic Eradication of Breast Tumors

Summary

Reversion of the malignant phenotype of erbB2-transformed cells can bedriven by anti-erbB2/neu monoclonal antibodies (mAb), which disrupt thereceptor's kinase activity. We examined the biologic effects of INF-γalone or after antierbB2/neu mAb treatment of erbB2-positive cells.IFN-γ had no effect on its own. Treatment of the tumors withanti-erbB2/neu mAb followed by IFN-γ led to dramatic inhibition of tumorgrowth in vitro and in vivo with minimal mAb dosing. Sequential therapyenhanced the effects of chemotherapy. Moreover, IFN-γ with mAb treatmentof mice with IFNγR knock down tumors did not demonstrate markedsynergistic eradication effects, indicating an unexpected role of INF-γon the tumor itself. Additionally, mAb and IFN-γ treatment also inducedimmune host responses that enhanced tumor eradication. Biochemicalanalyses identified loss of Snail expression in tumor cells, reflectingdiminution of tumor stem cell-like properties as a consequence ofaltered activity of GSK3-β and KLF molecules.

Significance

Targeting erbB2-driven tumors with mAb-based targeted therapy benefitspatient outcome in breast cancer; however, some patients do not respond,and virtually all responders eventually relapse. We have now found thatIFN-γ can modify intrinsic properties of transformed cells that haveundergone phenotypic reversion with anti-erbB2 mAb or lapatinib. Weestablish that IFN-γ concurrent and following anti-erbB2 mAb inhibitscertain intrinsic tumor-signaling pathways limiting stem cell-likeproperties. Sequential therapy synergistically provides optimal immunetherapeutic effects on erbB2-transformed human breast cancer cells andmouse erbB2-driven breast tumor models. Co-administration or sequentialordering of anti-erbB2 mAb with IFN-γ greatly reduces the amount of mAband genotoxic chemotherapeutics necessary for treatment of humans witherbB2-driven cancers.

Highlights

-   -   IFN-γ and 4D5 act directly on erbB2-positive breast cancer cells    -   IFN-γ, but not IFN-α or β, cooperates with 4D5 directly on        erbB2+ breast cancer cells    -   IFN-γ and 4D5 alters KLF4 levels and degrades Snail by the        GSK3-R/proteasome pathway    -   Sequential combination treatment with mAb and IFN-γ sensitizes        for tumor eradication

Introduction

The erbB or HER family of receptor tyrosine kinases consists of erbB1(the epidermal growth factor receptor (EGFR)/HER1), erbB2(p185/neu/HER2), erbB3 (HER3), and erbB4 (HER4), all of which can formhomomeric and heteromeric assemblies (Kokai et al., 1989; Qian et al.,1994b). These receptor tyrosine kinases participate in a variety ofsignal transduction cascades, including the Ras/Raf/MEK/ERK andPI-3K/Akt pathways. erbB2 is amplified in approximately 30% of breastcancer patients, and amplification is associated with poor prognosis anddecreased survival (Riemsma et al., 2012). In various cancers, amplifiedor mutated forms of these kinases drive increased proliferation,migration, survival, evasion of apoptosis, metastasis, and resistance tochemotherapeutics and ionizing radiation.

Recognition that mAbs could disable the p185 erbB2/HER2/neu tyrosinekinase receptor complex and also lead to reversal of the malignantphenotype challenged dogma that transformed cells could onlyprogressively become more abnormal (Drebin et al., 1985; Schechter etal., 1984). Reversal of the malignant phenotype by anti-erbB2 mAb beginsrapidly within 24 hours of mAb binding (Drebin et al., 1986; Lee et al.,2012; O'Rourke et al., 1997; Qian et al., 1994a) and occurs with downregulation of p185erbB2/neu receptor tyrosine kinase proteins causingdiminished enzymatic activity (Drebin et al., 1988a; Drebin et al.,1986; Furuuchi et al., 2007; Sliwkowski and Mellman, 2013; Wada et al.,1990; Zhang et al., 2007). These mechanistic events altering phenotypeoccur more dramatically with the inclusion of a second antibody, whichmore completely disables erbB2/neu kinase function (Drebin et al.,1988b; Furuuchi et al., 2007).

Tumor eradication that occurs in some partially syngeneic erbB2/neumodels also displayed a role for CD8+ T cells, macrophages and NaturalKiller cells (Park et al., 2010; Stagg et al., 2011). Cytokines derivedfrom CD8+ T cells and other cell types also contribute in certain tumormodels (Park et al., 2010; Stagg et al., 2011). IFN-γ, a cytokine thatplays diverse roles in innate and adaptive immune response, has beenimplicated in tumor immune responses. Stagg and colleagues demonstratedactivity of both type 1 and 11 IFNs in mediating anti-erbB2 mAbfunctions in vivo (Stagg et al., 2011) in non syngeneic tumor hostsystems.

Early biochemical studies indicated that IFN-γ could limit p185erbB2/neuexpression at the mRNA level (Marth et al., 1990) in some tumor lines.Conversely, IFN-γ alone was thought to increase erbB1 (EGFR) levels(Hamburger and Pinnamaneni, 1991) and TGFα secretion through increasedEGFR activity (Uribe et al., 2002) as well as to promote malignantgrowth of certain murine tumors (Beatty and Paterson, 2000). IFN-γ mayalso contribute to local environmental angiogenic effects (Coughlin etal., 1998). Historically, IFN-γ was one of the first recombinantcytokines tested as a single agent in trials of multiple human cancers,but led to few if any beneficial outcomes. Thus, clinical efforts usingIFN-γ as a primary single therapeutic for most malignancies have notbeen pursued (Krigel et al., 1985).

Certain proteins relevant to phenotypic developmental changes in stemcells and transformed cells have been described (Zheng and Kang, 2014).The transcriptional repressor Snail is essential for gastrulation andmesoderm formation during mammalian development (Carver et al., 2001).Snail levels increase in transformed cells. Elevated levels of Snailcontribute to tumor recurrence in vivo in erbB2/neu murine models andlevels of Snail may be relevant to relapse-free survival patterns inbreast cancer patients (Moody et al., 2005). Slug transcriptionalproteins may similarly function together to induce a stem-like phenotypein mammary cells in addition to maintaining tumor and metastaticproperties (Guo et al., 2012).

Glycogen synthase kinase 3-beta (GSK3-β), while negatively modified byAkt1, post-translationally regulates Snail through site-specificphosphorylation. Regulatory post-translational phosphorylationmodifications alter Snail's subcellular localization and stability.Specifically, GSK3-β phosphorylates Snail on six serine residues(serines 97, 101, 108, 112, 116, and 120) encompassing two motifs thatpromote translocation from the nucleus to the cytoplasm andβ-TRCP-mediated ubiquitination and degradation (Feinberg et al., 2005;Zhou et al., 2004). Zheng and Kang (Zheng and Kang, 2014) suggestedSnail effects phenotypic changes in cancer cells and describedepithelial to mesenchymal changes in both neu and ras transformed cells.

Products of activated immune cells such as IFN-γ have been shown toenhance the expression of the KLF4 transcription factor which itself canrepress Snail transcription (Feinberg et al., 2005; Yori et al., 2011).KLF4 over-expression induces macrophage activation markers while KLF4knockdown markedly modulates the ability of IFN-γ to render thoseeffects. Transfection of KLF4 attenuated primary tumor growth as well asaffecting development of metastatic lesions. erbB targeted immunetherapy processes that govern Snail protein functions have not beendescribed. Here we revealed that combinations of erbB2-targeted mAb andIFN-γ, but not IFN-α or β, can modify Snail expression and contribute tophenotypic reversion and cell viability by altering GSK3-β activity andKLF4 expression in breast tumors. We have also extended these efforts invivo in therapeutic and prevention models. Our findings provide a betterand less toxic human breast cancer therapeutic strategies.

Results

Sequential and Concurrent mAb 4D5 and IFN-γ Activities on erbB2+ BreastCancer Cells

erbB2 transformed human SK-BR-3 breast cancer cells were treated withIFN-γ and anti-erbB2 mAb (4D5) or control IgG (cIgG) at different times.Cells simultaneously exposed to both 4D5 and IFN-γ for eight days wereincluded. Pretreatment with 4D5 for 4 days followed by the addition ofIFN-γ caused a greater reduction in cell viability than pre-treatmentwith IFN-γ followed by addition of 4D5. Prolonged co-treatment with 4D5and IFN-γ resulted in greater reduction in cell viabilities (FIG. 54A).

Kinetics of the sensitizing effect were examined using an intermediatedose of IFN-γ (10 ng/mL). Dramatic reduction in cell viability was notedover an eight-day time course in the presence of 4D5, which could beaugmented with the inclusion of IFN-γ (FIG. 54B). These in vitrostudies, were performed with a single treatment of IFN-γ, 4D5, orIFN-γ+4D5 administered the day following cell seeding (i.e. Day 0).Immortalized, but untransformed MCF10A cells were refractory to 4D5alone or in combination with IFN-γ. These latter data indicate that thecombined ordered effects are manifest only on cells that have acquiredmalignant properties.

The effects of this treatment regimen were examined in prevention offoci formation in soft agar assays. Treatment with 4D5 produced adramatic reduction in the formation of foci in soft agar compared tocIgG and IFN-γ alone as demonstrated by a greater percentage of fociless than 20 pixels as well as fewer foci greater than 20. Addition ofIFN-γ produced a greater percentage of foci under 20 pixels than 4D5alone and a reduced percentage of foci greater than 20 pixels comparedto 4D5 alone (FIG. 54C and FIG. 54D).

Collectively, these studies established that disabling the p185erbB2kinase with mAb followed by IFN-γ produced the most significantphenotypic and viability effects on breast tumor cells.

Phenotypic Effects of mAb and INF-γ on Other Cell Types

We speculated that some erbB1 tumors might not be influenced byectodomain binding monoclonals, because of the action of IFN-γ onEGF/TGF expression (Uribe et al 2002) or by mutations which limit theeffects of targeting mAbs. U87 cells which are driven by EGFR VIII thatlacks portions of the extracellular domain of the EGFR, failed torespond to the anti-EGFR mAb C225. However, A431 cells, which lackerbB2, but express EGFR holoreceptor homomeric dimers similarlyresponded to dose-dependent increases of IFN-γ in the presence of C225(FIG. 55A) in a similar manner to erbB2/neu transformed cells.

The erbB2+ breast cancer cell lines MDA-MB-453 and BT-474 displaydistinct phenotypes from the SK-BR-3 cells. MDA-MB-453 and BT-474 wereinhibited by 4D5 mAb and MDA-MB-453 cell viability was further affectedby the inclusion of IFN-γ (FIG. 55B). Triple negative breast cancercells, MDA-MB-231, responded minimally to C225 mAb but also responded toIFN-γ alone (FIG. 55C). IFN-γ and a single mAb treatment did not haveadditive effects on BT-474 cell growth (FIG. 55D)

The PI3-K/Akt/GSK3-0 Snail Pathway can be Modified by mAb and IFN-γ orKinase Inhibitors Such as Lapatinib.

SK-BR-3 cells were treated with the dual EGFR/HER2 small moleculetyrosine kinase inhibitor lapatinib or 4D5 and C225 alone and incombination to determine which signaling pathways were affected bydisabling erbB family members. Lapatinib treatment inactivated both theAkt and MAPK pathways as anticipated. 4D5, but not C225, dominantlyinactivated Akt while not affecting MAPK activities. Combinations of 4D5and C225 to disable heteromeric kinase complexes, also inactivated Aktin SK-BR-3 cells. Disabling erbB2 in these cells activatedphosphorylated GSK3-β. We also found that treatment with 4D5, (butunexpectedly not with C225 alone), reduced Snail levels selectively.

We next compared the lapatinib response in SK-BR-3 and MDA-MB-453 cells.Lapatinib treatment diminished Akt activity in both cell lines and, to alesser extent, MAPK signaling. Lapatinib induced a dose-dependentreduction in Snail. Antagonism of PI3-kinase and AKT1/2 inhibitor inSK-BR-3 cells revealed a reduction in Snail, but not Slug.

We next determined if the combination of IFN-γ and anti-erbB2 mAb couldfurther reduce Snail protein levels. SK-BR-3 cells were treated withcIgG or 4D5 and increasing doses of IFN-γ. We found an IFN-γdose-dependent reduction of Snail only in the presence of 4D5 (FIG.56A). We found that GSK3-β activation is enhanced by IFN-γ in thepresence of 4D5 compared to IFN-γ treated samples in the presence ofcontrol IgG. Slug content was initially unaffected (FIG. 56A); however,by three days treatment led to reduction of Slug levels. Combination of4D5 and C225 in the presence of IFN-γ produced the most dramaticreduction in Snail and also reduced Slug content (FIG. 56B).

The notion that simultaneous mAb inhibition of HER2 and EGFR couldreduce Snail and Slug prompted us to examine whether small moleculekinase inhibitors such as lapatinib treatment could be modified byIFN-γ. SK-BR-3 cells treated with increasing doses of lapatinib andIFN-γ showed reduced Snail and Slug levels (FIG. 56C). Thus, erbBreceptors erbB2 and erbB1 (EGFR), functioning as heteromeric kinasesstabilize Snail and Slug, and combinatorial receptor disabling events inthe presence of IFN-γ provides optimal signal disruption and degradationof these proteins.

IFN a and IFN-0 do not Induce the Phenotypic Changes Seen with IFN-γ.

A recent study demonstrated that inclusion of IFN-β with an EGFRantibody produced a more potent anti-tumor effect than EGFR antibodyalone (Yang et al., 2014). Therefore, we compared IFN-β with IFN-γ inthe presence and absence of 4D5. Unexpectedly we found that IFN-βdramatically reduced Snail, even in the absence of 4D5 treatment (FIG.57A). Surprisingly, we found that IFN-β was cytotoxic to tumor cellseven at small doses in the absence of 4D5 (FIG. 57C). IFN-γ treatment ofMCF10A cells caused minimal changes to cell viability, whereas even lowdoses of IFN-β were cytotoxic to these cells (FIG. 57B). In vivo studiesalso failed to determine effects of IFN-α and anti-erbB2 mAb (data notshown). Therefore, we believe that under these conditions only IFN-γacts to complete phenotypic reversion engendered by anti-erbB2 mAbtherapy.

Reduction of Snail Protein is Triggered by Elevated GSK3-β and OccursPrimarily Through Altered Proteasome-Mediated Degradation

Snail is predominantly degraded through the proteasome and, to a lesserextent, the lysosome. We initially tested the role of GSK3-β using thesmall molecule inhibitor. Inhibition of GSK3-β in the presence of theco-treatment regimen resulted in a dose-dependent rescue of Snail butproduced no change in Slug content (FIG. 58A). Proteasome inhibitorMG-132 inhibited the co-treatment mediated reduction of Snail in adose-dependent manner, also with no change in Slug content (FIG. 58B).Conversely, inhibition of lysosomal function with chloroquine was notable to rescue the 4D5 and IFN-γ-mediated degradation of Snail.Therefore, we conclude that 4D5 and IFN-γ-mediated Snail degradation ismediated primarily through the proteasome.

Next we examined direct effect of snail decrease by this orderedtreatment in GSK3-β knockdown cells. Although GSK3-β was knockdowned inSK-BR3 cells by shRNA vectors, snail expression was still significantlydecreased.

We consider other pathways are also affected by snail functions. Moraland colleagues (Moral et al., 2009) reported that KLF4 expressionpatterns were increased in tumor samples from mice with hyper-activatedAkt. We examined the effects of ordered and combined targeting antibodyand IFN-γ on KLF4 levels in SK-BR-3 cells stably transfected with GSK3-βshRNA. The ability to alter Snail functions was directly linked to KLF4levels and GSK3-β.

Since treatment with 4D5 and IFN-γ activated GSK3-β and active GSK3-βpromotes Snail degradation, expression of a version of Snail with theGSK3-β phosphorylation sites mutated to alanines should prevent thisdegradation. We transiently expressed empty vector (EV), HA-tagged Snailwild type (WT) (Kajita et al., 2004), and HA-tagged Snail with serines97, 101, 108, 112, 116, and 120 mutated to alanine (6SA) (Zhou et al.,2004) in SK-BR-3 cells. Exogenous WT Snail was degraded whereas 6SASnail was largely resistant to 4D5 and IFN-γ-induced proteasomaldegradation in the nuclear fraction (FIG. 58C). In cells transfectedwith the EV control, we observed Snail in the cytoplasm only when thecells were treated with both 4D5 and IFN-γ (FIG. 58C) indicating thatboth treatments are required for Snail translocation to the cytoplasm.In addition, Snail knock down in SK-BR-3 cells increases the effects of4D5 targeted therapy on anchorage dependent cell growth patterns. Theseresults indicate that snail degradation that are promoted by 4D5 plusIFN-γ ordered treatment occurs through the GSK3-R/proteasome pathway,and decreasing Snail levels are important for targeted therapy.

Effects of mAb and IFN-γ In Vivo: Ordered Combinations Reduce the Needfor Large Amounts of Targeting mAb

mAb 7.16.4 is biologically active in vivo and in vitro. In vitro mAb7.16.4 is active against cells transformed with the rat or humanerbB2/neu oncogene and disables the p185erbB2/neu kinase leading todiminished downstream p185erbB2/neu signaling (Drebin et al., 1986;Zhang et al., 1999). We as well as others examined various optimizeddoses of anti-erbB2/neu mAb therapy. Five mg/kg of 7.16.4 mAb have beenused intravenously (Drebin et al., 1986) and/or on every other dayintraperitoneally in therapeutic studies of tumor growth (Du et al.,2013; Stagg et al., 2011).

We examined if reduced amounts of mAb could be used if IFN-γ wasprovided after therapy was initiated. As shown in FIG. 59A, whenentirely syngeneic MMTV-neu transgenic mice were treated with asub-optimal dose of 7.16.4 (1.5 mg/kg), the mAb was unable tosubstantially inhibit the growth of H2N113 tumors. IFN-γ treatment alonealso failed to significantly inhibit tumor growth. However, thecombination of suboptimal amounts of 7.16.4 and IFN-γ completelyarrested the growth of H2N113 tumors. Importantly, the pattern of thedata in FIG. 59A was reminiscent of the in vitro tumor cellproliferation kinetics in FIG. 59B. Finally, histologic examination ofthe tumor tissues after treatment revealed significant necrosis only inmice treated with both 7.16.4 and IFN-γ.

To examine if IFN-γ was targeted to tumor cells directly or to hostelements in the vicinity of the tumor, we limited the expression of theIFN-γ receptor on these tumor cells. Using shRNA, we created an IFN-γreceptor knockdown species of the H2N113 cell line: H2N113 (IFNγR KD)(FIG. 59B). The reduced expression level of IFN-γ receptor was confirmedin several distinct ways. IFNγRKD tumor cells form progressively growingtumors. Nevertheless treatment with suboptimal mAb doses generated onlya modest growth reduction of IFNγRKD tumor growth (FIG. 59B). IFN-γ hadno effect on its own against tumors that had diminished IFN-γ receptorlevels.

Ordered Therapy Also Affects Myeloid Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) are phenotypically CD45+, CD11b+and GR-1+. Co-treatment with sub optimal 7.16.4 and IFN-γ led toreduction of MDSC populations invading tumor tissues (FIG. 59C). Wenoted that there were limited but comparable numbers of Foxp3 cells incontrol and treated animals.

Since we have determined that both mAb and IFN-γ must interfere withtumor cells directly, we evaluated chemo-attractant effects of moleculeselaborated by tumor cells on MDSC that might be affected by the combinedtreatment. MDSC isolated from the spleen of tumor-bearing mice wereplaced into the apical chamber of a transwell system. Conditioned mediumfrom H2N113 cells treated directly with mAb, IFN-γ or their combinationswere tested for their ability to attract MDSC cells to migrate into thebasolateral chamber. As shown in FIG. 59D, 7.16.4 alone slightly reducedthe tumor-promoted migration, but cotreatment of the tumor cells with7.16.4 and IFN-γ blocked MDSC migration. IFN-γ alone had no appreciableeffects.

Enhanced Role for IFN Type II Signals in erbB Cytolytic ImmuneResponses.

Several researches suggested a role for T cells (Park et al., 2010;Stagg et al., 2012; Stagg et al., 2011) in some component of the hostresponse to 7.16.4 monoclonal antibody. We used suboptimal mAb and IFN-γin syngeneic systems and collected effector CD8+ T cells from eachtreatment group to investigate their activity against H2N113 cells. Thecombination therapy group showed modest but definite effector T cellsactivity against p185erbB2/neu positive tumors. IFN-γ alone hadnegligible effects. These data support the notion that minor antigensbecome more relevant when we use mAb and IFN-γ ordered therapy.

Macrophages may contribute to the effector response. Two types offunctional macrophages have been proposed to infiltrate the tumormicroenvironment and such invasion is promoted by molecules elaborateddirectly or indirectly by tumor cells. We examined tumors for invadingantiinflammatory, pro-tumor M2 and pro-inflammatory, cytolyticanti-tumor M1 types. mAb 7.16.4 and IFN-γ increased M1 cell accumulationwithin adjacent areas of the tumor, and animals treated with thiscombination therapy had the highest M1 frequency.

Based on these data we suggest that immune regulation is affectedindirectly by the dominant effect of mAb and IFN-γ on the tumor itself.Ordered therapy reduces tissue and immune regulatory cell activity inthe microenvironment and this reduction is permissive for effector Tcells to act on phenotype reversed tumor cells.

In Vivo Evidence of Modification of Stem Cell Characteristics by mAb andIFN-γ.

Molecular analyses in vitro revealed that the ordered treatment ofbreast tumor cells with anti-erbB2/neu mAb and IFN-γ diminishedexpression of Snail proteins, which mediates stem cell like properties.ALDH1 expression patterns also define cancer stem cell phenotypes, andits expression correlates with breast cancer prognostic features(Douville et al., 2009; Ginestier et al., 2007). We compared tumors atthe end of treatment for ALDH1 levels. Co-treatment with 7.16.4 andIFN-γ treatment reduced the expression of ALDH1 in p185erbB2/neu tumors(FIG. 59E).

Growing tumor cells were also studied for expression patterns of Snail.We noted a reduced level of Snail protein in tumors undergoingeradication as a consequence of mAb and IFN-γ therapy in vivo (FIG.59E). These studies directly correlate with in vitro findings describedin FIG. 54A-FIG. 54D examining anchorage dependent and independenteffects of combinations of anti erbB mAb and IFN-γ.

Ordered mAb and IFN-γ Therapy can Prevent Breast Cancer Tumorigenesisand Synergistically Inhibits Tumor Growth with Chemotherapy.

To determine if these therapeutic effects were relevant to prevention oftumor development (adjuvant use of this combination) we chose to examinea model we have previously described, using small tumor inocula. Weexamined the effects of treatment of MMTV-neu female mice withcombinations of 7.16.4 and 7.9.5 mAb, with or without IFN-γ, in animalsimplanted with small tumor inocula to mimic incipient tumors. We noted adramatic reduction when IFN-γ was incorporated in the treatmentprotocol. As can be seen in FIG. 59F, we were able to limit tumor growthin this prevention model with minimal amounts of dual targeting mAb.These studies indicate that animals treated with ordered therapy of mAband IFN-γ can mount potent intrinsic cytotoxic elimination of smallnumbers of incipient tumor cells. These data support previous studiesshowing a delayed emergence of tumors when mAb specific for erbB2/neuwas used as an adjuvant therapy in mouse prevention studies (Finkle etal., 2004;

Katsumata et al., 1995) To extend these studies to a clinical context,we added docetaxel in subtherapeutic quantities to evaluate if potenttumor inhibition with small amounts of phenotype reversing mAb wouldalso limit the required genotoxic amounts of currently employedchemotherapy (FIG. 59G). Treatment with 7.16.4 followed by IFN-γ anddocetaxel led to inhibition of tumor growth compared with other groupsdespite using suboptimal doses of both anti erbB antibody andchemotherapy. Our conclusion is that optimizing phenotype reversionrepresents a critical element the evolution of precision drug therapy ofbreast carcinoma.

IFN-γ Dramatically Promotes Tumor PD-L1 Expression and which Representsa Target for Tumor Therapy.

We observed aggressive tumor growth with IFN-γ alone treated mice. Thisis also consistent with why IFN-γ alone is not useful for cancer therapy(Zaidi and Merlino, 2011). Stagg and colleagues showed treatment withIFN-γ induces PDL1 expression (Stagg et al., 2011). It is possible thatPD-L1 acts as a negative immune regulator induced by IFN-γ treatmentalone. As shown in FIG. 60A, IFN-γ dramatically induced PD-L1 expressionin IFN-γ treated tumor tissues. FACS analysis showed this up-regulationalso occurs in tumor cells themselves (FIG. 60B). To determine if PD-L1can be further targeted for this ordered therapy, we used anti-PD-L1antibody along with 7.16.4 and IFN-γ. As expected, administration ofanti-PD-L1 antibody with 7.16.4 and IFN-γ caused the most significanttumor regression in those groups (FIG. 60C). These results indicate thatPD-L1 represents an additional target when used with mAb and IFN-γordered therapy.

Discussion

Therapeutics that target p185erbB2/HER2/neu are effective forrestraining human malignant disease but are rarely curative. Disablingof the p185erbB2/neu kinase complex leads to phenotypic reversal ofmalignant properties, but is incomplete and subject to furthermodification (Drebin et al., 1986; Lee et al., 2012; O'Rourke et al.,1997; Wada et al., 1990). This phenotypic state is more sensitive togenotoxic damage by chemotherapeutic and radiation effects, or immunemediated lytic processes. Our studies identified a second transitionprocess that occurs after mAb-mediated down-regulation of p185erbB2/neuproteins from the cell surface, which can be induced by IFN-γ. Thistransition involves a role of transcription factors such as Snail knownto be relevant to cancer stem cell features. IFN-γ alone, without thefirst reversion step is ineffective in being able to induce a malignantphenotype reversion or change in Snail levels.

We describe a dramatic and almost complete arrest of tumor growth evenwith suboptimal doses of anti-erbB2/neu mAb when IFN-γ is included andin vivo. Because IFN-γ enhances antibody effects at very low doses, thisapproach may improve targeted therapy effectiveness in tissues whereonly low levels of antibody might penetrate. Dramatic tumor inhibitoryeffects can be also accomplished with minimal amounts of targeting mAband IFN-γ followed by chemotherapeutics (docetaxel). These studiesidentify a benefit of this rational approach to precision medicine thatwill be accompanied by lessened toxicity during tumor treatment.

The effect of mAb and IFN-γ are a consequence of phenotypic reversalchanges on the tumor itself as well as effects of IFN-γ on hostelements. The synergistic effects of mAb and IFN-γ were lost when tumorcells with reduced IFN-γ receptor levels were targeted. At themicroenvironment level, we noticed a reduction of regulatory cells thatlimit immune reactions by suppressing functions of immune cells. Tumorelaborated chemo-attractants which recruit regulatory cells werediminished upon ordered treatment of tumor cells with mAb and thenIFN-γ. We have been unable to document any comparable phenotypicactivity in the well established models we use, when IFN-α or IFN-β wereused (in vivo or in vitro respectively) rather than IFN-γ.

Contributions of regulatory MDSC cells in the local tumor environmentmay be important in limiting immune elimination of breast tumors andtheir activity is diminished with ordered mAb and IFN-γ therapy.Enhanced accumulation of M1 type macrophages in the local tumorenvironment in situations using combined mAb and IFN-γ therapy wereobserved. Modest enhancement of cytolytic T cells active against erbB2tumors was also noted. Together these data support an observablecontributory role for the immune system in targeted therapy and a rolefor IFN-γ in enhancing immune eradication of erbB tumors that haveundergone phenotype reversal. ALDH1 is diminished in tumor cells treatedin vivo with combined mAb and IFN-γ therapy. Elevation of ALDH1 enzymelevels correspond to erlotinib resistance in erbB1 (EGFR) mutated humantumors (Corominas-Faja et al., 2013). Thus metabolic changes occurwithin tumor cells with stem cell like properties, that are amenable tomanipulation by phenotypic reversion best promoted by anti-p185erbB2/neumAb and by IFN-γ. Consequently, tumor tissues should be examineddiagnostically for expression of IFN-γ receptor and aldolase insituations in which targeted therapy might be administered.

IFN-γ is a cytokine which is considered to activate anti-tumor immunitywith its cytostatic/cytotoxic activities. However, there are few IFN-γdefined beneficial outcomes clinically when used alone. Thus, clinicalefforts using IFN-γ as a primary single therapeutic for mostmalignancies have not been pursued (Krigel et al., 1985). We found, ashas been reported by others that IFN-γ treatment increases PD-L1expression in the tumor tissues and cells. The PD-1/PD-L1 pathways alongwith CTLA-4 represent elements of a checkpoint pathway and contribute toregulating tumor immunity (Ott et al., 2013). Interestingly the factthat PD-L1 expression is increased by IFN-γ in tumor site/cells may berelevant to the success of ordered therapy. Combining targeted therapywith immune therapy in an ordered way can lead to a greatly reduced needfor the mAb components and the genotoxic chemotherapeutics needed totreat humans with erbB caused cancers.

Experimental Procedures

Lysate Preparation and Western Blotting

Cells were lysed in a RIPA-based buffer with protease inhibitors (RocheDiagnostics) and used for Western blot.

MTT Assays

Cells were seeded and treated the following day (Day 0). Treated cellswere grown for the indicated times; and on the day of the assay MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagentwas added and values were determined by absorbance at 570 nm on a platereader (Tecan).

Soft Agar Growth Assays

Plates were coated with agarose mixed with DMEM. Cells were overlaid onthe bottom layer in an agarose (final agarose concentration of 0.2%)DMEM mixture supplemented with the indicated therapeutic treatment.After two weeks, viable foci were visualized with MTT reagent. Each wellwas imaged using an Alpha Imager, and foci size was determined usingNIH-endorsed ImageJ software.

Mouse In Vivo Experiments

6-10 weeks old female MMTV-neu mice were used for this set ofexperiments. For the in vivo treatment model, a rodent erbB2/neutransformed Balb/c breast tumor cell line, H2N113 (1×106), was injectedsubcutaneously into both sides of the back of mice. Tumor size wasmeasured with a digital caliper and calculated using a simple algorithm(3.14×length×wide×height+6.)

Flow Cytometry

At 1 day after final treatment, spleens and tumors were collected andsingle cell suspensions made. Cell surface antigens were stained withthe antibodies indicated. Cells were analyzed with FACS LSR (BDBiosciences) and FACS data were analyzed with FlowJo software (TreeStar, Ashland, Oreg.).

MDSC Migration Assay

To prepare conditioned medium, H2N113 cells were seeded and cultureduntil sub-confluent in culture. Cells were then treated with 7.16.4and/or IFN-γ at indicated concentrations for 3 days. Then thesupernatants were collected for the migration assay. Migration of MDSCwas measured by the Transwell system (pore size: 4 μm, Corning). After 3hr incubations at 37° C., the cells that migrated to bottom layers werecollected and subjected to flow cytometry.

Statistical Consideration

When applicable, statistical analysis was performed by Student's t-testusing Microsoft Excel. At a minimum, data with a p-value <0.05 weredeemed significant. In all cases, experiments shown are representativesthat were performed at least twice.

Discussion

Unlike non-transformed replicating cells which can be killed by exposureto therapeutic radiation or chemotherapy, tumor cells are resistant toinduction of cell death by radiation and chemotherapy. It has beendiscovered that by disrupting the multimeric ensembles which produceelevated kinase activity associated with the transformed phenotype of acancer cell, such a cancer cell, which is ordinarily resistant toradiation or chemotherapy induced cell death, becomes sensitive toradiation. Accordingly, one aspect of the present invention providesmethods of making radiation or chemotherapy-resistant cancer cellsradiation or chemotherapy-sensitive. The present invention relates tomethods of treating an individual who has cancer cells that havemultimeric receptor ensembles comprising p185her2/neu or EGFR whichprovide kinase activity associated with a transformed phenotype. Themethod comprises the step of first administering to the subject acomposition that disrupts the kinase activity associated with themultimeric receptor ensemble. The composition may also inhibit theattraction of immune suppressor cells by cancer cells. The subject isthen treated with radiation or a chemotherapeutic agent.

There are several known receptor ensembles which, in cancer cells,display elevated kinase activity that is associated with the transformedphenotype. Members of the erbB family of receptors are known to formmultimeric ensembles which result in elevated tyrosine kinase activityin tumor cells. Multimeric ensembles involving erbB family membersinclude erbB homodimers as well as erbB heterodimers comprisingmonomeric components from different erbB family members. Multimericreceptor ensembles of platelet derived growth factor receptors (PDGFR)also display elevated kinase activity that is associated with thetransformed phenotype.

The present invention is useful to therapeutically treat an individualidentified as suffering from erbB-associated tumors, such asneu-associated tumors, in order to reverse the transformed phenotype ofthe tumor cells. The present invention is useful to prophylacticallytreat an individual who is predisposed to develop erbB-associated tumorsor who has had erbB-associated tumors and is therefore susceptible to arelapse or recurrence.

According to one aspect of the present invention, dimer formation oferbB proteins in erbB mediated cancer cells is disrupted to render suchcells more susceptible to cell destruction using radiation orchemotherapy. The ability of the cancer cells to attract immunesuppressor cells may also be disrupted to render such cells moresusceptible to cell destruction using radiation or chemotherapy.Accordingly, combination therapies are provided that comprise firstadministering to an individual a composition which comprises an activeagent that results in interference of erbB dimerization, andinterferon-gamma, followed by exposing the subject to therapeuticamounts of radiation or administering to the patient a therapeuticamount of a chemotherapeutic agent. According to these aspects of theinvention, methods for treating individuals who have an erbB proteinmediated tumor are provided.

In some tumor cells, the p185her2/neu translation product of c-erbB2gene is overexpressed and forms homodimers and heterodimers with othererbB family members. Such dimerization of overexpressed p185her2/neuleads to elevated tyrosine kinase activities which is associated withthe transformed phenotype. Disruption of tyrosine kinase activity, suchas by inhibiting dimer formation between monomeric components, resultsin a cytostatic effect on the tumor cells. The disruption also rendersthe previously resistant cancer cells radiation-sensitive andchemoptherapy-sensitive.

Similarly, in some tumor cells, a mutant form of EGFR (ΔEGFR) isexpressed which is ligand-independent. ΔEGFR forms homodimers andheterodimers with wild-type EGFR and other erbB family members. Suchdimerization of ΔEGFR leads to elevated tyrosine kinase activities whichis associated with the transformed phenotype. Disruption of tyrosinekinase activity, such as by inhibiting dimer formation between monomericcomponents, results in a cytostatic effect on the tumor cells. Thisdisruption also renders the previously resistant cancer cellsradiation-sensitive and chemoptherapy-sensitive.

In some embodiments, the erbB-protein mediated tumor is a brain cancertumor. In some preferred embodiments, the erbB-protein mediated tumor isa glial tumor. In some preferred embodiments, the erbB-protein mediatedtumor is a glioblastoma. In some embodiments, the erbB-protein mediatedtumor is a breast cancer tumor. In some embodiments, the erbB-proteinmediated tumor is an ovarian cancer tumor. In some embodiments, theerB-protein mediated tumor is a pancreatic cancer tumor.

As described and exemplified herein, IFN-gamma (IFNγ) in combinationwith an antibody against p185her2/neu leads to a much more effectivetreatment for erbB2/Her2/neu cancers than IFN-gamma or the antibody whenadministered singly. The present invention provides clinicalapplications using currently available IFN-gamma together with anti-ErbBantibodies (e.g, Herceptin, Pertuximab, or Erbitux, etc).

The activity of IFNγ in combination with an anti-p185her2/neu or ananti-EGFR antibody is profoundly enhanced. The significant effectdescribed herein is surprising.

When administered singly, IFNγ has no significant activity. The examplesherein show that it has little or no effect on the proliferation ofcancer cells. As a monotherapy, IFNγ is not effective for treatingsubjects afflicted with cancer. Surprisingly, the combination of IFNγwith an anti-ErbB antibody has enhanced activity on cancer cells.Additionally, the combination of IFNγ with an anti-p185her2/neu antibodyor an anti-EGFR antibody increases the sensitivity of cancer cells toradiation therapy and chemotherapy.

In Example 2, IFNα appears to have anti-tumor activity when administeredas a monotherapy in an in vivo tumor model, but the combination of IFNαand an anti-p185her2/neu antibody does not have significantly enhancedactivity. Without wishing to be bound by any scientific theory, itappears that IFNα behaves differently from IFNγ when administered as amonotherapy or in combination with an anti-p185her2/neu antibody.

Aspects of the present invention relate to the surprising discovery thatthe combination of IFNγ with an anti-p185her2/neu or an anti-EGFRantibody has profoundly enhanced activity, and has enhanced efficacy fortreating cancer. In some embodiments the combination of IFNγ with ananti-p185her2/neu or an anti-EGFR antibody increases sensitivity ofcancer cells to a chemotherapeutic agent or radiation.

The action of the 4d5 construct on tumor cells in vitro, as shown in theExamples, suggests that a dominant phenotype is induced with alteredgrowth and reduced snail expression. In some embodiments, there is anintrinsic stem cell like phenotype that is inhibited by the actions ofIFNγ after the malignant phenotype is reversed by disabling the receptorkinase. Without wishing to be bound by any scientific theory, an ordereddisabling needed; first with the anti-p185her2/neu or an anti-EGFRantibody or with a kinase inhibitor of p185her2/neu or EGFR, resultingin a phenotype which is more normalized, i.e. less malignant. Theresultant phenotype which is more normalized can then be acted on by theIFNγ. The IFNγ disables the stem cell like features of this morenormalized transformed cell. Accordingly, aspects of the invention areuseful both for the prevention of tumor development, and for cancertreatment.

Without wishing to be bound by any scientific theory, in someembodiments the intent of treatment is to sensitize cells by conversionof phenotype. In the first event the anti-p185her2/neu or an anti-EGFRantibody disables the p185her2/neu or EGFR kinase making the cellsamenable to further phenotypic change by IFNγ which renders them moreable to be damaged by genotoxic signals.

Snail and Slug Data Shown in FIG. 11 -FIG. 13

Epithelial to mesenchymal transition (EMT) is believed to be a criticaldeterminant of breast cancer progression. Cells that have undergone EMTshare features of cancer stem cells (Mani et al., 2008) such asresistance to therapy (Korkaya et al., 2012) and evasion of the hostimmune system (Akalay et al., 2013). EMT is mediated by a group oftranscription factors including Snail, Slug, Twist, and ZEB1, which arecollectively known as the master regulators of EMT. Snail is awell-established critical determinant of breast cancer EMT andmetastasis and its regulation at the post-translational level isbelieved to be critical to breast cancer progression. Therefore,treatment strategies that accelerate and/or maintain the degradation ofSnail or Slug (or both) would be adventitious because they would reversethe phenotype of a dedifferentiated or stem-like cell to a moredifferentiated cell. The biological significance of this is these cellswould then be more sensitive to genotoxic insults.

Aspects of the present invention relates to the discovery thatco-administration of IFNγ and an anti-ErbB antibody accelerates and/ormaintains the degradation of Snail or Slug (or both) in cells that arenot malignant. Some embodiments of the present invention relate to firsttreating a subject with IFNγ and an anti-ErbB antibody or a fusionprotein of the invention to increase the sensitivity of cancer cells,and of cells that have undergone EMT, to chemotherapy or radiation, andthen administering chemotherapy or radiation to the subject.

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1. A method of treating a p185her2/neu-associated tumor in a subjectcomprising administering to the subject a therapeutically effectiveamount of a first anti-p185her2/neu antibody or an antigen bindingportion thereof, a second anti-p185her2/neu antibody or an antigenbinding portion thereof, and interferon-gamma (IFNγ).
 2. The method ofclaim 1, wherein the first anti-p185her2/neu antibody or an antigenbinding portion thereof and the second anti-p185her2/neu antibody or anantigen binding portion thereof bind to distinct epitopes of the p185ectodomain.
 3. The method of claim 1, wherein the firstanti-p185her2/neu antibody is trastuzumab.
 4. The method of claim 3,wherein the second anti-p185her2/neu antibody is pertuzumab.
 5. Themethod of claim 1, wherein the first anti-p185her2/neu antibody ispertuzumab.
 6. The method of claim 1, comprising administering to thesubject a therapeutically effective amount of a radiation or achemotherapeutic agent.
 7. The method of claim 6, wherein thechemotherapeutic agent comprises a taxane.
 8. The method of claim 7,wherein the taxane is paclitaxel.
 9. The method of claim 1, wherein thep185her2/neu-associated tumor is breast cancer.
 10. A method ofprophylactically treating a subject who is predisposed to developing ap185her2/neu-associated tumor comprising administering to the subject atherapeutically effective amount of a first anti-p185her2/neu antibodyor an antigen binding portion thereof, a second anti-p185her2/neuantibody or an antigen binding portion thereof, and interferon-gamma(IFNγ).
 11. The method of claim 10, wherein the first anti-p185her2/neuantibody or an antigen binding portion thereof and the secondanti-p185her2/neu antibody or an antigen binding portion thereof bind todistinct epitopes of the p185 ectodomain.
 12. The method of claim 10,wherein the first anti-p185her2/neu antibody is trastuzumab.
 13. Themethod of claim 12, wherein the second anti-p185her2/neu antibody ispertuzumab.
 14. The method of claim 10, wherein the firstanti-p185her2/neu antibody is pertuzumab.
 15. The method of claim 10,comprising administering to the subject a therapeutically effectiveamount of a radiation or a chemotherapeutic agent.
 16. The method ofclaim 15, wherein the chemotherapeutic agent comprises a taxane.
 17. Themethod of claim 16, wherein the taxane is paclitaxel.
 18. The method ofclaim 10, wherein the p185her2/neu-associated tumor is breast cancer.19. The method of claim 10, wherein the subject is at risk of relapse orrecurrence of the p185her2/neu-associated tumor.
 20. The method of claim10, wherein the method delays the development of thep185her2/neu-associated tumor for a period of at least 30 days.